Rapamycin inhibits B-cell activating factor (BAFF)-stimulated cell proliferation and survival by suppressing Ca²⁺-CaMKII-dependent PTEN/Akt-Erk1/2 signaling pathway in normal and neoplastic B-lymphoid cells

Abstract

B-cell activating factor (BAFF) is a crucial survival factor for B cells, and excess BAFF contributes to development of autoimmune diseases. Recent studies have shown that rapamycin can prevent BAFF-induced B-cell proliferation and survival, but the underlying mechanism remains to be elucidated. Here we found that rapamycin inhibited human soluble BAFF (hsBAFF)-stimulated cell proliferation by inducing G₁-cell cycle arrest, which was through downregulating the protein levels of CDK2, CDK4, CDK6, cyclin A, cyclin D1, and cyclin E. Rapamycin reduced hsBAFF-stimulated cell survival by downregulating the levels of anti-apoptotic proteins (Mcl-1, Bcl-2, Bcl-xL and survivin) and meanwhile upregulating the levels of pro-apoptotic proteins (BAK and BAX). The cytostatic and cytotoxic effects of rapamycin linked to its attenuation of hsBAFF-elevated intracellular free Ca²⁺ ([Ca²⁺]_i). In addition, rapamycin blocked hsBAFF-stimulated B-cell proliferation and survival by preventing hsBAFF from inactivating PTEN and activating the Akt-Erk1/2 pathway. Overexpression of wild type PTEN or ectopic expression of dominant negative Akt potentiated rapamycin’s suppression of hsBAFF-induced Erk1/2 activation and proliferation/viability in Raji cells. Interestingly, PP242 (mTORC1/2 inhibitor) or Akt inhibitor X, like rapamycin (mTORC1 inhibitor), reduced the basal or hsBAFF-induced [Ca²⁺]_i elevations. Chelating [Ca²⁺]_i with BAPTA/AM, preventing [Ca²⁺]_i elevation using EGTA, 2-APB or verapamil, inhibiting CaMKII with KN93, or silencing CaMKII strengthened rapamycin’s inhibitory effects. The results indicate that rapamycin inhibits BAFF-stimulated B-cell proliferation and survival by blunting mTORC1/2-mediated [Ca²⁺]_i elevations and suppressing Ca²⁺-CaMKII-dependent PTEN/Akt-Erk1/2 signaling pathway. Our finding underscores that rapamycin may be exploited for prevention of excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.

1. Introduction

The B-cell activating factor from the TNF family (BAFF), also known as BLyS, THANK,TALL-1, and zTNF4, a type II membrane protein that exists in both membrane-bound and soluble forms, is known to be crucial for proliferation and differentiation of B lymphocytes [1; 2; 3; 4]. BAFF triggers proliferation and survival signals via three receptors: BAFF-R (BR3), BCMA and TACI [4; 5; 6; 7]. Normal level of BAFF plays a critical role in maintaining normal humoral immunity, whereas excessive BAFF causes immune system disorders [6; 8]. Mounting studies have demonstrated that excessive BAFF is the culprit of malignant B-cell proliferation [9; 10], and increased levels of BAFF are involved in a number of autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and SjÖgren’s syndrome (SS) [8; 11; 12; 13].

Rapamycin is not only a lipophilic macrolide antibiotic but also a specific mTOR inhibitor [14]. A series of recent studies have shown that rapamycin is effective in the treatment of murine and human SLE [15; 16; 17]. Our group has demonstrated that pretreatment with rapamycin potently inhibits hsBAFF-stimulated cell proliferation and survival by suppressing mTOR-mediated PP2A-Erk1/2 signaling pathway in normal and neoplastic B-lymphoid cells [18]. However, whether rapamycin executes the action against excessive BAFF-induced B-cell proliferation and survival through other mechanisms needs to be further explored.

Intracellular calcium ion (Ca²⁺) acts as a second messenger responsible for numerous cellular events, such as proliferation/growth, differentiation, and survival in various immune cells [19; 20]. Calcium/calmodulin-dependent protein kinase II (CaMKII), a ubiquitously expressed multifunctional serine/threonine kinase, functions through Ca²⁺ signaling to regulate the development and activity of many different cell types including immune cells [21; 22; 23]. Upon binding to Ca²⁺/calmodulin (CaM), CaMKII is activated by autophosphorylation [24]. Studies have demonstrated that the activation of MEK-Erk1/2 in B lymphocytes is dependent on calcium influx [22; 25]. Activated CaMKII is involved in regulating the activation of Erk1/2 leading to myeloid leukemia cell proliferation [21]. Recently, we have shown that hsBAFF-stimulated B-cell proliferation and survival by Ca²⁺-CaMKII-mediated mTOR and Erk1/2 network [26; 27]. However, it is unclear whether rapamycin inhibits BAFF-stimulated B-cell proliferation and survival through Ca²⁺-CaMKII-mediated Erk1/2 signaling.

The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a phosphatase, which dephosphorylates phosphatidylinositol 3,4,5-trisphosphate to phosphatidylinositol 3,4-bisphosphate, thereby negatively regulating the phosphoinositide 3-kinase (PI3K) pathway [28; 29]. Expression of PTEN inhibits proliferation and survival in PTEN-mutant tumor cells [30; 31]. Emerging evidence has shown that PTEN can also negatively regulate the Erk1/2 pathway, which contributes to inhibition of tumor development [32]. The pathology of autoimmune diseases relates to the reduced expression/activity of PTEN [33; 34]. For example, decreased PTEN expression was associated with SLE development [33]. In high-invasive fibroblast-like synoviocytes of RA patients, PTEN expression is reduced or hard to detect [34]. In addition, PI3K/Akt may activate Erk1/2 through PKC [32]. Based on the above findings, we postulated that rapamycin may mediate a cross-talk between PTEN, Akt and Erk1/2 pathways via Ca²⁺-CaMKII-dependent mechanism in B cells in response to BAFF.

Here, we show that rapamyicn inhibits BAFF-stimulated B-cell proliferation and survival by blunting mTORC1/2-mediated [Ca²⁺]_i elevations and suppressing Ca²⁺-CaMKII-dependent PTEN/Akt-Erk1/2 signaling pathway. The results enhance our understanding of the molecular mechanisms by which rapamycin exerts prevention against excessive BAFF-induced aggressive B cell malignancies and autoimmune diseases.

2. Materials and methods

2.1. Reagents

Anti-CD19 magnetic fluorobeads-B was purchased from One Lambda (Canoga Park, CA, USA). Akt inhibitor X and PP242 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Refolded human soluble BAFF (hsBAFF) was a recombinant form of the extracellular domain of the BAFF produced in Escherichia coli from this group [35]. RPMI 1640 medium and fetal bovine serum (FBS) were supplied by Gibco (Rockville, MD, USA). CellTiter 96®AQ_ueous One Solution Cell Proliferation Assay kit was from Promega (Madison, WI, USA). Enhanced chemiluminescence solution was from Sciben Biotech Company (Nanjing, China). 1,2-bis(oAminophenoxy) ethane-N,N,N’,N’-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA/AM) and 2-aminoethoxydiphenyl borane (2-APB) were purchased from Calbiochem (San Diego, CA, USA), whereas ethylene glycol tetra-acetic acid (EGTA) was purchased from Sigma (St. Louis, MO, USA). ML-SA1 was supplied by Abcam Technology (Cambridge, UK). Verapamil hydrochloride was from MedChemExpress (Monmouth Junction, NJ, USA). Rapamycin and KN93 were from ALEXIS (San Diego, CA, USA), while U0126 was from Sigma. Other chemicals were purchased from local commercial sources and were of analytical grade.

2.2. Cells

Neoplastic B-lymphoid Raji and Daudi cell lines (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin at 37°C in a humidified incubator containing 5% CO₂. Primary B lymphocytes were purified from fresh splenic cells of healthy mice using anti-CD19 magnetic fluorobeads and cultured as described previously [26]. All procedures used in this study were approved by the Institutional Animal Care and Use Committee, and were in compliance with the guidelines set forth by the Guide for the Care and Use of Laboratory Animals.

2.3. Recombinant adenoviral constructs and infection of cells

Recombinant adenovirus expressing wild-type human PTEN (Ad-PTEN), and the control adenovirus expressing the green fluorescent protein (GFP) (Ad-GFP) were described previously [36; 37]. Recombinant adenovirus encoding HA-tagged dominant negative Akt (Ad-dn-Akt, T308A/S473A) was generously provided from Dr. Kenneth Walsh (Boston University, Boston, MA). For experiments, Raji cells were grown in the growth medium and infected with the individual adenovirus for 24 h at 5 of multiplicity of infection (MOI = 5). Subsequently, the cells were used for experiments. Cells infected with Ad-GFP served as a control. Expression of PTEN and HA-tagged dn-Akt was detected by Western blotting with antibodies to PTEN and HA, respectively.

2.4. Lentiviral shRNA cloning, production and infection of cells

Lentiviral shRNAs to CaMKII and GFP (for control) were constructed and infected as described previously [36; 38].

2.5. Cell proliferation and viability assay

Purified mouse B lymphocytes, Raji and/or Daudi cells, or Raji cells infected with Ad-PTEN, Ad-dn-Akt and Ad-GFP, respectively, or Raji cells infected with lentiviral shRNAs to CaMKII and GFP, respectively, were seeded in 24-well plates (3×10⁵ cells/well, for cell proliferation assay) or 96-well plates (3×10⁴ cells/well, for cell viability assay) and cultured for overnight in a humidified incubator of 5 % CO₂ at 37°C. The next day, cells were pretreated with/without rapamycin (100 ng/ml) for 2 h or Akt inhibitor X (20 μM) or U0126 (5 μM) for 1 h, or pretreated with/without rapamycin for 2 h and then with/without BAPTA/AM (20 μM), EGTA (10 μM), 2-APB (100 μM) or KN93 (10 μM) for 1 h, or pretreated with/without ML-SA1(10 μM) or verapamil (10 μM) for 10 h and then with/without rapamycin for 2 h, followed by stimulation with/without hsBAFF (1 and/or 2.5 μg/ml) for 48 h with 5 replicates of each treatment. Subsequently, cell number was counted using a Coulter Counter (Beckman Coulter, Fullerton, CA, USA), and cell viability was measured with a Victor X3 Light Plate Reader (PerkinElmer, Waltham, MA, USA), as described previously [18].

2.6. Live cell evaluation by trypan blue exclusion

Purified mouse B lymphocytes, Raji and Daudi cells were seeded in 24-well plates (3 ×10⁵ cells/well). The next day, cells were treated with/without hsBAFF (1 and 2.5 μg/ml) for 48 h following pre-incubation with/without rapamycin (100 ng/ml) for 2 h. Then, live cells were recorded by counting viable cells using trypan blue exclusion.

2.7. [Ca²⁺]_i detection

Purified mouse B lymphocytes, Raji and/or Daudi cells were seeded in 6-well plates at a density of 2×10⁶ cells/well under standard culture conditions and kept overnight at 37°C humidified incubator with 5%CO₂. The next day, after treatment, the cells were harvested and washed three times with PBS, followed by dilution to 2×10⁶ cells/ml with PBS. Subsequently, cell suspensions (100 μl) for [Ca²⁺]_i analysis were loaded with 20 μl of 5 μM Fluo-3/AM for 40 min at 37°C in the dark, and then washed three times with PBS to remove the extracellular Fluo-3/AM. PBS, replacing Fluo-3/AM, served as a negative control. Finally, the cells for each example were re-suspended with 1 ml PBS to detect status of [Ca²⁺]_i by a fluorescence-activated cell sorter (FACS) Vantage SE flow cytometer (Becton Dickinson, California, USA), or by excitation at 488 nm and emission at 535 nm for adding the suspension into a 96-well plate (150 μl/well) using a Victor X3 Light Plate Reader.

2.8. Cell cycle analysis

Raji and Daudi cells were seeded in 6-well plates at a density of 2×10⁶ cells/well and cultured as described above. The next day, cells were treated with/without hsBAFF (1 and 2.5 μg/ml) for 48 h following pre-incubation with/without rapamycin (100 ng/ml) for 2 h, followed by a brief wash with PBS. Cell suspensions were centrifuged at 1000 rpm for 5 min, and the pellets were fixed with pre-chilled (−20°C) absolute ethanol and stained with the Cell Cycle Analysis Kit (Vazyme Biotech Company, Nanjing, China). Percentages of cells within each of the cell cycle compartments (G₀/G₁, S, or G2/M)) were recorded with a FACS Vantage SE flow cytometer and analyzed using ModFit LT software (Verity Software House, Topsham, ME). Cells treated with vehicle alone (PBS) served as a control.

2.9. Western blot analysis

Purified mouse B lymphocytes, Raji and/or Daudi cells, or Raji cells infected with Ad-PTEN, Ad-dn-Akt and Ad-GFP, respectively, or Raji cells infected with lentiviral shRNAs to CaMKII and GFP, respectively, after treatments, were subjected to Western blotting as described [39]. The following antibodies were used: CaMKII, phospho-CaMKII (Thr286), phospho-Erk1/2 (Thr202/Tyr204), phospho-Akt (Ser473), phospho-Akt (Thr308) (Cell Signaling Technology, Danvers, MA, USA), CDK2, CDK4, CDK6, cyclin A, cyclin D1, cyclin E, Cdc25A, Cdc25B, p21, p27 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-PTEN (Thr366), PTEN (Epitomics, Burlingame, CA, USA), β-actin, survivin, Bcl-2, Akt, HA (Sciben Biotech Company)；Goat anti-rabbit IgG-horseradish peroxidase (HRP), goat anti-mouse IgG-HRP, and rabbit anti-goat IgG-HRP (Pierce, Rockford, IL, USA).

2.10. Statistical analysis

Results were expressed as mean values ± standard error (mean ± SE). Analysis of statistical significance was performed using Student’s t-test for non-paired replicates. Group variability and interaction were compared using either one-way or two-way ANOVA followed by Bonferroni’s post-test to compare replicate means. Significance was accepted at P < 0.05.

3. Results

3.1. Rapamycin attenuation of hsBAFF-elevated [Ca²⁺]_i links to its inhibition of proliferation and survival in normal and lymphoma B cells

We have recently shown that hsBAFF elevates intracellular free Ca²⁺ ([Ca²⁺]_i) level, contributing to B-cell proliferation/viability [26; 27], and rapamycin inhibits hsBAFF-stimulated proliferation and survival [18] in Raji cells, Daudi cells and/or purified mouse B lymphocytes. Here, we hypothesized that rapamycin’s inhibition of hsBAFF-induced B-cell proliferation and survival may be related to attenuation of hsBAFF-induced [Ca²⁺] elevation. To test this hypothesis, first of all, [Ca²⁺]_i was measured, using an intracellular Ca²⁺ indicator dye, Fluo-3/AM, by the manifestation of fluorescence intensity under a microplate reader. Our time course (0–12 h) experiments showed that stimulation with hsBAFF (1 and 2.5 μg/ml) for 8–12 h was able to significantly elicite [Ca²⁺]_i rises in Raji cells, Daudi cells and purified mouse B lymphocytes (Fig.1A). In line with our previous reports [26; 27], a higher [Ca²⁺]_i level was observed in the cells stimulated with hsBAFF for 12 h (Fig. 1A). So, this time point was selected for more studies, as described below.

Fig. 1.

Rapamycin attenuates hsBAFF-induced [Ca²⁺]_i elevation as well as proliferation and survival in normal and lymphoma B cells. Raji cells, Daudi cells and purified mouse splenic B lymphocytes were stimulated with/without hsBAFF (1 and 2.5 μg/ml) for 0–12 h, or pretreated with/without rapamycin (Rapa, 100 ng/ml) for 2 h and then stimulated with/without hsBAFF (1 and 2.5 μg/ml) for 12 h (for [Ca²⁺]_i detection) or 48 h (for cell proliferation and viability assays). (A-C) [Ca²⁺]_i manifestation was detected by a microplate reader (A, B) or FACS (C) using an intracellular Ca²⁺ indicator dye Fluo-3/AM. (D) Quantitative analysis of [Ca²⁺]_i fluorescence intensity by FACS assay. (E) Cell proliferation was estimated by cell counting. (F) Cell viability was determined by MTS assay. (G) The relative number of live cells was evaluated by trypan blue exclusion assay. Results are presented as mean ± SE (n = 3–6). ^aP<0.05, difference with control group; ^bP<0.05, difference with 1 μg/ml hsBAFF group, ^cP<0.05, difference with 2.5 μg/ml hsBAFF group.

As expected, pretreatment with rapamycin (100 ng/ml) significantly attenuated hsBAFF-elevated [Ca²⁺]_i in the cells (Fig.1B). In addition, our flow cytometry also confirmed that rapamycin suppressed hsBAFF-elevated level and staining of [Ca²⁺]_i in a larger population of Raji cells, Daudi cells and purified mouse B lymphocytes (Fig. 1C and D). Furthermore, consistent with our previous findings [18], rapamycin reduced hsBAFF-stimulated cell proliferation and survival, as evaluated by cell counting (Fig. 1E), MTS assay (Fig. 1D), and trypan blue exclusion (Fig. 1G), respectively. Taken together, these data suggest that rapamycin attenuation of hsBAFF-elevated [Ca²⁺]_i links to its inhibition of proliferation and survival in normal and lymphoma B cells.

3.2. Rapamycin inhibits hsBAFF-stimulated B-cell proliferation and survival by downregulating the activity of G₁/S-CDKs and reducing the ratio of anti-apoptotic/pro-apoptotic proteins

It is known that cell proliferation is associated with cell cycle progression [40], and rapamycin inhibits proliferation of malignant B cells by altering the expression of key regulatory proteins related to G₁/S cell cycle progression [41; 42]. To understand how rapamycin inhibits hsBAFF-stimulated B-cell proliferation, cell cycle analysis was performed. Raji and Daudi cells were pretreated with rapamycin (100 ng/ml) for 2 h and then treated with hsBAFF (1 and 2.5 μg/ml) for 48 h, followed by PI staining and flow cytometry. As shown in Fig. 2A–C, rapamycin inhibited hsBAFF-stimulated cell cycle progression by increasing cell population at G₀/G₁ phase and concurrently decreasing cell population at S phase. Next, to unveil the mechanism behind this, we examined the expression of cyclin-dependent kinases (CDKs) and related regulatory proteins, including cyclins, Cdc25 and CDK inhibitors (p21^Cip1 and p27^Kip1) in the cells. The results showed that rapamycin did not apparently alter the protein levels of Cdc25A, Cdc25B, p21^Cip1, and p27^Kip1, but markedly downregulated the protein levels of CDK2, CDK4, CDK6, cyclin A, cyclin D1, and cyclin E in the cells treated with or without hsBAFF (Fig. 2D). As rapamycin can trigger apoptosis by causing imbalance of anti-apoptotic/pro-apoptotic proteins [43; 44; 45], next, we examined whether rapamycin reduces hsBAFF-stimulated cell survival through this mechanism. At First, an experiment of time course (0–12 h) for the effect of hsBAFF on expressions of Bcl-2-family proteins was conducted in Raji cells and purified mouse B lymphocytes. The results showed that hsBAFF (2.5 μg/ml) obviously increased the levels of anti-apoptotic proteins (Mcl-1, Bcl-2, Bcl-xL and survivin) and meanwhile decreased the levels of pro-apoptotic proteins (BAK and BAX) at 8–12 h in the cells (Fig. 2E), consistent with the finding at the time at which hsBAFF-induced [Ca²⁺]_i elevations (Fig. 1A). Next, we revealed that rapamycin downregulated the anti-apoptotic proteins and upregulated the pro-apoptotic proteins in the cells in response to hsBAFF (Fig. 2F). Collectively, these results suggest that rapamycin inhibits hsBAFF-stimulated B-cell proliferation and survival by downregulating the activity of G₁/S-CDKs and reducing the ratio of anti-apoptotic/pro-apoptotic proteins.

Fig. 2.

Rapamycin inhibits hsBAFF-stimulated B-cell cycle progression and cell viability by downregulating cyclins, CDKs, and anti-apoptotic proteins and upregulating pro-apoptotic proteins. Raji cells, Daudi cells and/or purified mouse splenic B lymphocytes were stimulated with/without hsBAFF (2.5 μg/ml) for 0–12 h, or pretreated with/without rapamycin (Rapa, 100 ng/ml) for 2 h and then stimulated with/without hsBAFF (1 and 2.5 μg/ml) for 12 h (for Western blotting) or 48 h (for cell cycle analysis). (A) Histograms from a representative experiment showing the effect of rapamycin and/or hsBAFF on cell cycle profile in B cells. (B, C) The distribution of cells at G₀/G₁ and S phases of the cell cycle was quantified, respectively. (D-F) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-actin as a loading control. Similar results were observed in three independent experiments. Results are presented as mean ± SE (n = 6). ^aP<0.05, difference with control group; ^bP<0.05, difference with 1 μg/ml hsBAFF group, ^cP<0.05, difference with 2.5 μg/ml hsBAFF group.

3.3. Rapamycin inhibits hsBAFF-stimulated B-cell proliferation and viability by preventing hsBAFF from inactivating PTEN and activating the Akt-Erk1/2 signaling pathway

It has been shown that PTEN may negatively regulate Erk1/2 pathway in several malignancies, and Akt may activate Erk1/2 through PKC [32]. We recently have demonstrated that rapamycin inhibits BAFF-stimulated B-cell proliferation and survival by suppressing Erk1/2 pathway [18]. Here, we tested whether rapamycin blocks hsBAFF-induced Erk1/2 activation and proliferation/viability by modulating PTEN-Akt signaling in B cells. Interestingly, pretreatment with rapamycin (100 ng/ml) for 2 h potently prevented hsBAFF (1 and 2.5 μg/ml)-induced phosphorylation of PTEN in Raji cells, Daudi cells and purified mouse B lymphocytes (Fig. 3A), implying that rapamycin blocks hsBAFF inactivation of PTEN. Subsequently, Raji cells, infected with recombinant adenovirus expressing wild-type human PTEN (Ad-PTEN) or Ad-GFP (as control), were pretreated with/without rapamycin (100 ng/ml) for 2 h or Akt inhibitor X (20 μM) for 1 h, followed by stimulation with hsBAFF (2.5 μg/ml) for 12 h or 48 h. The results showed that the infection with Ad-PTEN increased the expression of PTEN and decreased the basal and hsBAFF-induced phosphorylation of Akt and Erk1/2, as well as protein levels of Bcl-2 and survivin, compared to the infection with Ad-GFP (Fig. 3B). Overexpression of PTEN potentiated the inhibitory effects of rapamycin or Akt inhibitor X on hsBAFF-induced p-Akt and p-Erk1/2, Bcl-2 and survivin (Fig.3B). Of note, overexpression of PTEN also enhanced rapamycin’s or Akt inhibitor X’s suppression of hsBAFF-stimulated B-cell proliferation and survival (Fig. 3C and D). The findings support the notion that rapamycin inhibits hsBAFF-induced activation of Erk1/2 and consequential B-cell proliferation/viability, by preventing hsBAFF inactivation of PTEN and activation of Akt.

Fig. 3.

Rapamycin suppresses hsBAFF-stimulated B-cell proliferation and viability by targeting the PTEN/Akt-Erk1/2 signaling pathway. Raji cells, Daudi cells and purified mouse splenic B lymphocytes, or Raji cells infected with Ad-PTEN, Ad-dn-Akt and Ad-GFP (for control), respectively, were pretreated with/without rapamycin (Rapa, 100 ng/ml) for 2 h, or Akt inhibitor X (20 μM) or U0126 (5 μM) for 1 h, followed by stimulation with/without hsBAFF (1 and/or 2.5 μg/ml) for 12 h (for Western blotting) or 48 h (for cell proliferation and viability assays). (A, B, E) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-actin as a loading control. Similar results were observed in at least three independent experiments. (C, F) Cell proliferation was evaluated by cell counting. (D, G) Cell viability was determined by MTS assay. Results are presented as mean ± SE (n = 6). ^aP<0.05, difference with control group; ^bP<0.05, difference with 2.5 μg/ml hsBAFF group; ^cP<0.05, Ad-PTEN group or Ad-dn-Akt group vs Ad-GFP group.

To further verify the role of Akt in rapamycin’s blockage of hsBAFF-induced Erk1/2 activation and proliferation/viability in B cells, recombinant adenovirus expressing HA-tagged dominant negative Akt (Ad-dn-Akt) was employed. As shown in Fig. 3E, a high level of HA-tagged Akt mutant was seen in Raji cells infected with Ad-dn-Akt, but not in the cells infected with Ad-GFP (as control). Ectopic expression of dn-Akt markedly repressed hsBAFF-triggered p-Akt, p-Erk1/2, Bcl-2 and survivin (Fig.3E). Rapamycin, but not U0126, strongly suppressed hsBAFF-stimulated p-Akt. However, both rapamycin and U0126 substantially inhibited hsBAFF-induced p-Erk1/2, Bcl-2, and survivin (Fig. 3E). Of importance, expression of dn-Akt was able to strengthen the inhibitory effects of rapamycin or U0126 on hsBAFF-induced p-Akt, p-Erk1/2, Bcl-2 and survivin (Fig. 3E), as well as proliferation/viability (Fig. 3F and G) in the cells. These data indicate that rapamycin inhibits hsBAFF-stimulated B-cell proliferation/viability by preventing hsBAFF from inactivating PTEN and activating the Akt-Erk1/2 signaling pathway.

3.4. Rapamycin suppresses hsBAFF-mediated PTEN/Akt-Erk1/2 signaling in a Ca²⁺-dependent manner

Having observed that rapamycin’s inhibition of hsBAFF-elevated [Ca²⁺]_i was involved its suppression of B-cell proliferation and survival (Fig. 1), and rapamycin prevented hsBAFF-induced B-cell proliferation/viability by regulating the PTEN/Akt-Erk1/2 signaling pathway (Fig. 3), this prompted us to investigate the mechanisms by which rapamycin blunts hsBAFF-induced [Ca²⁺]_i elevations, and whether rapamycin inhibition of hsBAFF-elevated [Ca²⁺]_i plays a pivotal role in repressing hsBAFF-inactivated PTEN/-activated Akt signaling, thereby leading to activation of Erk1/2 and proliferation/viability in B cells. Firstly, mTORC1/2 inhibitor PP242 and Akt inhibitor X were employed to evaluate the contribution of mTORC2, mTORC1 and Akt in hsBAFF-induced [Ca²⁺]_i elevations in Raji cells and purified mouse B lymphocytes. The results showed that pretreatment with PP242 or Akt inhibitor X, like that of rapamycion, potently declined the basal or hsBAFF-induced [Ca²⁺]_i elevations (Fig. 4A), implying an upstream role for mTORC1/2 in mediating hsBAFF-induced [Ca²⁺]_i elevations. Next, Raji cells and purified mouse B lymphocytes were pretreated with/without rapamycin (100 ng/ml) for 2 h and then treated with/without BAPTA/AM (20 μM), an intracellular Ca²⁺ chelator, for 1 h, followed by exposure to hsBAFF (2.5 μg/ml) for 12 h or 48 h. We found that treatment with rapamycin or BAPTA/AM alone decreased the basal or hsBAFF-triggered [Ca²⁺]_i elevation, and the combination of rapamycin with BAPTA/AM more potently attenuated hsBAFF-triggered [Ca²⁺]_i elevation in the cells (Fig. 4B). Furthermore, co-treatment with rapamycin and BAPTA/AM showed more powerful inhibitory effects on hsBAFF-elicited phosphorylation of PTEN, Akt and Erk1/2 than treatment with hsBAFF or BAPTA/AM alone in the cells (Fig. 4C). The inhibitory effects of BAPTA/AM on hsBAFF-induced Bcl-2 and survivin were also remarkably potentiated by co-treatment with rapamycin (Fig. 4C). In line with this, BAPTA/AM obviously suppressed hsBAFF-induced proliferation and viability in Raji cells and purified mouse B lymphocytes, and these effects were strengthened by rapamycin as well (Fig. 4D and E). The results suggest that rapamycin’s intervention in hsBAFF-mediated PTEN/Akt-Erk1/2 signaling pathway and B-cell proliferation/viability is Ca²⁺-dependent.

Fig. 4.

Rapamycin disturbs the PTEN/Akt-Erk1/2 signaling pathway and B-cell proliferation/viability by inhibiting hsBAFF-induced [Ca²⁺]_i elevation. Raji cells and purified mouse splenic B lymphocytes were pretreated with/without rapamycin (Rapa, 100 ng/ml) for 2 h and/or Akt inhibitor X (20 μM), PP242 (1 μM) or BAPTA/AM (20 μM) for 1 h, followed by stimulation with/without hsBAFF (2.5 μg/ml) for 12 h (for [Ca²⁺]_i detection and Western blotting) or 48 h (for cell proliferation and viability assays). (A, B) [Ca²⁺]_i manifestation was detected by a microplate reader using an intracellular Ca²⁺ indicator dye Fluo-3/AM. (C) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-actin as a loading control. Similar results were observed in at least three independent experiments. (D) Cell proliferation was evaluated by cell counting. (E) Cell viability was determined by MTS assay. Results are presented as mean ± SE (n = 6). ^aP<0.05, difference with control group; ^bP<0.05, difference with 2.5 μg/ml hsBAFF group; ^cP <0.05, difference with hsBAFF/Rapa group or hsBAFF/BAPTA/AM group.

To further unveil how rapamycin attenuation of hsBAFF-elevated [Ca²⁺]_i contributes to its disruption of PTEN/Akt-Erk1/2 signaling pathway and B-cell proliferation/viability, we extended our studies using EGTA (100 μM), an extracellular Ca²⁺ chelator, 2-APB, an inhibitor for both inositol 1,4,5-trisphosphate (IP₃) receptors endoplasmic reticulum (ER) and the Ca²⁺ release activated Ca²⁺ (CRAC) channels, ML-SA1, an agonist of TRPML1 channel for lysosomal Ca²⁺ release [46], or verapamil, a blocker of TRPML1 channel [46]. The results showed that inhibition of extracellular Ca²⁺ influx by EGTA, or preventing hsBAFF-induced [Ca²⁺]_i elevation using 2-APB or verapamil (Fig. 5A and 6A) profoundly prevented hsBAFF from eliciting the phosphorylation of PTEN, Akt and Erk1/2 (Fig. 5B), and substantially blocked the upregulation of Bcl-2 and survivin, as well as proliferation/viability in Raji cells and purified mouse B lymphocytes in response to hsBAFF (Fig. 5B–D and 6B–D). Of importance, co-treatment with rapamycin/EGTA, rapamycin/2-APB or rapamycin/verapamil resulted in a more effective inhibition of hsBAFF-elicited the events (Fig. 5A–D and 6A–D). In addition, we also observed that treatment with ML-SA1 alone markedly triggered the elevation of [Ca²⁺]_i, p-PTEN, p-Akt, p-Erk1/2, Bcl-2, survivin and proliferation/viability, and there existed further increases after stimulation with hsBAFF in the presence of ML-SA1, which were repressed by rapamycin pretreatment in the cells (Fig. 6A–D). These findings indicate that rapamycin’s interference with hsBAFF-induced extracellular Ca²⁺ influx, ER Ca²⁺ release and lysosomal Ca²⁺ release links to its suppression of the PTEN/Akt-Erk1/2 signaling pathway and B-cell proliferation/viability. Collectively, our data strongly support the notion that rapamycin blocks hsBAFF-induced B-cell proliferation/viability via preventing hsBAFF elevation of [Ca²⁺]_i, thus hindering the PTEN/Akt-Erk1/2 signaling pathway.

Fig. 5.

Rapamycin hinders the PTEN/Akt-Erk1/2 signaling pathway and B-cell proliferation/viability through preventing hsBAFF-induced extracellular Ca²⁺ influx and ER Ca²⁺ release. Raji cells and purified mouse splenic B lymphocytes were pretreated with/without rapamycin (Rapa, 100 ng/ml) for 2 h and then with/without EGTA (100 μM) or 2-APB (100 μM) for 1 h, followed by stimulation with/without hsBAFF (2.5 μg/ml) for 12 h (for [Ca²⁺]_i detection and Western blotting) or 48 h (for cell proliferation and viability assays). (A) [Ca²⁺]_i manifestation was detected by a microplate reader using an intracellular Ca²⁺ indicator dye Fluo-3/AM. (B) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-actin as a loading control. Similar results were observed in at least three independent experiments. (C) Cell proliferation was evaluated by cell counting. (D) Cell viability was determined by MTS assay. Results are presented as mean ± SE (n = 6). ^aP<0.05, difference with control group; ^bP<0.05, difference with 2.5 μg/ml hsBAFF group; ^cP<0.05, difference with hsBAFF/Rapa group, hsBAFF/EGTA group or hsBAFF/2-APB group.

Fig. 6.

Rapamycin affects the PTEN/Akt-Erk1/2 signaling pathway and B-cell proliferation/viability via blocking hsBAFF-induced lysosomal Ca²⁺ release. Raji cells and purified mouse splenic B lymphocytes were pretreated with/without ML-SA1 (10 μM) or verapamil (10 μM) for 10 h and then with/without rapamycin (Rapa, 100 ng/ml) for 2 h, followed by stimulation with/without hsBAFF (2.5 μg/ml) for 12 h (for [Ca²⁺]_i detection and Western blotting) or 48 h (for cell proliferation and viability assays). (A) [Ca²⁺]_i manifestation was detected by a microplate reader using an intracellular Ca²⁺ indicator dye Fluo-3/AM. (B) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-actin as a loading control. Similar results were observed in at least three independent experiments. (C) Cell proliferation was evaluated by cell counting. (D) Cell viability was determined by MTS assay. Results are presented as mean ± SE (n = 6). ^aP<0.05, difference with control group; ^bP<0.05, difference with 2.5 μg/ml hsBAFF group; ^cP <0.05, difference with hsBAFF/Rapa group, hsBAFF/ML-SA1 group or hsBAFF/Verapamil group.

3.5. Rapamycin diminishes hsBAFF-stimulated B-cell proliferation and viability by blocking Ca²⁺-CaMKII-dependent PTEN/Akt-Erk1/2 signaling pathway

CaMKII is a universal integrator of Ca²⁺ signaling [47; 48]. In response to the binding of the Ca²⁺/calmodulin complex, CaMKII is activated by autophosphorylation at several sites, including Thr286, Thr305, and Thr306 [21; 49]. Thus, we further investigated whether rapamycin prevents hsBAFF-induced B-cell proliferation/viability by inhibiting Ca²⁺-CaMKII-dependent PTEN/Akt-Erk1/2 signaling pathway. For this, firstly, Raji cells and purified mouse B lymphocytes were pretreated with/without rapamycin (100 ng/ml) for 2 h and then stimulated with/without hsBAFF (1 and 2.5 μg/ml) for 12 h, followed by Western blot analysis. As shown in Fig. 7A, pretreatment with rapamycin dramatically reversed hsBAFF-triggered CaMKII phosphorylation. This was in agreement with the observation that there existed a significant inhibitory effect of rapamycin on hsBAFF-induced B-cell proliferation and survival in Raji cells and purified mouse B lymphocytes (Fig. 1E–G). The results imply that rapamycin may attenuate B-cell proliferation and survival by preventing hsBAFF from activating CaMKII.

Fig. 7.

Rapamycin inhibits hsBAFF-stimulated B-cell proliferation and viability through suppressing Ca²⁺-CaMKII-dependent PTEN/Akt-Erk1/2 signaling pathway. Raji cells and purified mouse splenic B lymphocytes, or Raji cells infected with lentiviral shRNA to CaMKII or GFP (control), were pretreated with/without rapamycin (Rapa, 100 ng/ml) for 2 h, or pretreated with/without BAPTA/AM (20 μM), EGTA (100 μM), 2-APB (100 μM) or KN93 (10μM) for 1 h post pre-incubation with/without Rapa for 2 h, followed by stimulation with/without hsBAFF (2.5 μg/ml) for 12 h (for Western blotting) or 48 h (for cell proliferation and viability assays). (A-C, F, G) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-actin as a loading control. Similar results were observed in at least three independent experiments. (D, H) Cell proliferation was evaluated by cell counting. (E, I) Cell viability was determined by MTS assay. Results are presented as mean ± SE (n = 6). ^aP<0.05, difference with control group; ^bP<0.05, difference with 2.5 μg/ml hsBAFF group; ^cP<0.05, difference with hsBAFF/Rapa group or hsBAFF/KN93 group; ^dP < 0.05, CaMKII shRNA group vs GFP shRNA group.

Next, we investigated whether rapamycin’s suppression of hsBAFF-induced activation of CaMKII depends on the level of [Ca²⁺]_i. To address this question, Raji cells and purified mouse B lymphocytes were pretreated with/without rapamycin (100 ng/ml) for 2 h and then BAPTA/AM (20 μM), 2-APB (100 μM) or EGTA (100 μM) for 1 h, followed by stimulation with/without hsBAFF (2.5 μg/ml) for 12 h. As predicted, chelating [Ca²⁺]_i with BAPTA/AM or preventing [Ca²⁺]_i elevation using 2-APB or EGTA markedly potentiated rapamycin’s inhibition of hsBAFF-increased CaMKII phosphorylation in the cells (Fig. 7B), suggesting that rapamycin prevents hsBAFF-elevated [Ca²⁺]_i, leading to inhibition of CaMKII phosphorylation. To clarify whether rapamycin’s inhibition of CaMKII phosphorylation is implicated in affecting the PTEN/Akt-Erk1/2 signaling pathway and proliferation/viability in B cells in response to hsBAFF, Raji cells and purified mouse B lymphocytes were pretreated with/without rapamycin (100 ng/ml) for 2 h and then treated with/without CaMKII inhibitor KN93 (10 μM) for 1 h, followed by stimulation with hsBAFF (2.5 μg/ml) for 12 h or 48 h. The results showed that KN93 substantially enhanced the inhibitory effects of rapamycin on hsBAFF-elicited p-CaMKII, p-PTEN, p-Akt and p-Erk1/2 in the cells (Fig. 7C). Also, co-treatment with rapamycin/KN93 prevented cells from hsBAFF-evoked Bcl-2, survivin and proliferation/viability more potently than rapamycin or KN93 alone (Fig. 7C–E). To further corroborate the role and significance of CaMKII in rapamycin’s inhibition of hsBAFF-induced events in B cells, Raji cells, infected with lentiviral shRNA to CaMKII or GFP, were treated with/without hsBAFF (2.5 μg/ml) for 12 h or 48 h following pretreatment with/without rapamycin (100 ng/ml) for 2 h. As shown in Fig. 7F, CaMKII expression was downregulated by ~90% in shRNA CaMKII-infected cells compared to shRNA GFP-infected cells. Knockdown of CaMKII reinforced rapamycin suppression of hsBAFF-induced phosphorylation of CaMKII, PTEN, Akt and Erk1/2 in the cells (Fig. 7G). Consistently, silencing CaMKII also enhanced the inhibitory effects of rapamycin on hsBAFF-induced Bcl-2 and survivin, and proliferation/viability in the cells (Fig. 7G–I). Taken together, these data validate that rapamycin reduced hsBAFF-stimulated B-cell proliferation and viability by blocking Ca²⁺-CaMKII-dependent PTEN/Akt-Erk1/2 signaling pathway.

4. Discussion

B-cell activating factor of the TNF family is critical for B-cell proliferation, maturation, and survival, and excessive BAFF is linked to aggressive/neoplastic B-cell disorders, including RA, SS, and SLE [4; 50; 51]. It has been reported that excessive endogenous or transgenic BAFF in mice prolongs the life span and increases the population of peripheral B lymphocytes [9; 50]. Patients suffer from a great quantity of autoantibodies secreted from aggressive B cells, whose root is excess BAFF [50; 52]. A lot of evidence points to an important role of excess BAFF-evoked aggressive or neoplastic B-cell disorders in the pathophysiology of autoimmune diseases [52; 53]. However, so far we have no effective options to prevent the development of the BAFF-induced disorders. Therefore, it is necessary to find a novel therapeutic target and strategy to control excess BAFF-promoted B-cell expansion.

Rapamycin is a specific mTOR inhibitor, which is widely used in organ transplantation by inhibiting T-cell activation [14]. Recent studies have shown that rapamycin is an effective treatment in murine lupus models and the patients with human SLE, shedding new light on the role of rapamycin in the treatment of SLE [54; 55; 56]. It has been described that rapamycin could directly alter molecular abnormalities in SLE T cells related to calcium signaling [16; 54]. Recently we have identified that rapamycin prevents BAFF-stimulated B-cell proliferation and survival by suppressing mTOR-mediated PP2A-Erk1/2 signaling pathway [18]. Herein, we provide evidence that rapamycin effectively prevented excessive hsBAFF-induced [Ca²⁺]_i elevation, thereby suppressing CaMKII-dependent PTEN/Akt-Erk1/2 signaling pathway and proliferation/survival in normal and neoplastic B-lymphoid cells.

Ca²⁺ signaling is an important component of signal transduction pathways regulating B and T lymphocyte proliferation and survival [21]. Our recent studies have revealed that hsBAFF-upregulated [Ca²⁺]_i promotes the proliferation/viability in Raji cells and purified mouse B lymphocytes [26; 27]. Rapamycin inhibits hsBAFF-stimulated B-cell proliferation and survival [18]. In this study, we showed that rapamycin’s prevention of hsBAFF-elevated [Ca²⁺]_i linked to its inhibition of proliferation and survival in Raji cells, Daudi cells and purified mouse B lymphocytes (Fig. 1). Further investigation found that rapamycin inhibited hsBAFF-stimulated B-cell proliferation (Fig. 1) by arresting the cells at G0/G1 phase of the cell cycle (Fig. 2A–C). It has been reported that BAFF could upregulate Mcl-1, Bcl-2 and Bcl-xL, downregulate Bax, enhance the synthesis of cyclin D2, and activate NF-κB, as well as CDK4 in normal and neoplastic B-lymphoid cells [57; 58; 59]. Rapamycin causes cell proliferation reduction and apoptosis by disturbing expression of Mcl-1, Bcl-2, Bcl-xL and Bax [43; 44; 45], as well as expression of CDKs and related regulatory proteins, including cyclins, Cdc25 and CDK inhibitors (p21^Cip1 and p27^Kip1) related to the G1/S cell cycle progression in multiple cell types including normal and malignant B cells [40; 41; 42]. In the current study, we noticed that rapamycin substantially downregulated the protein levels of CDK2, CDK4, CDK6, cyclin A, cyclin D1, and cyclin E, but had no obvious effect on the levels of Cdc25A, Cdc25B, p21^Cip1 and p27^Kip1, in Raji cells, Daudi cells and purified mouse B lymphocytes treated or untreated with hsBAFF (Fig. 2D). Rapamycin also downregulated the levels of anti-apoptotic proteins (Mcl-1, Bcl-2, Bcl-xL and survivin) and meanwhile upregulated the levels of pro-apoptotic proteins (BAK and BAX) in the cells stimulated by hsBAFF. Chelating [Ca²⁺]_i with BAPTA/AM mimicked the effect of rapamycin, suppressing hsBAFF-induced Bcl-2 and survivin, cell proliferation and viability (Fig.4). Taken together, our data imply that rapamycin hinders hsBAFF-induced [Ca²⁺]_i elevation, thereby inhibiting hsBAFF-stimulated B-cell proliferation and survival.

PTEN, a well-known tumor suppressor, has been documented as a negative regulator of Akt signaling [60; 61]. We observed that rapamycin rescued PTEN activity and inhibited Akt, as rapamycin attenuated hsBAFF-induced phosphorylation of PTEN and Akt in Raji cells and purified mouse B lymphocytes (Fig. 3A and B). Recent data have pointed out that PTEN also negatively regulates Erk1/2 pathway, and Akt is able to activate Erk1/2 through PKC in several malignancies [32]. Putting all data together, we reasoned that there exists a cross-talk between PTEN, Akt and Erk1/2 pathways in B cells stimulated by hsBAFF, which is mediated by rapamycin. Here, for the first time, we present evidence that rapamycin prevented hsBAFF from inactivation of PTEN and activation of the Akt-Erk1/2 pathway, leading to reduced cell proliferation/viability in B cells. This is strongly supported by the findings that overexpression of PTEN or dn-Akt or pretreatment with Akt inhibitor X or U0126 dramatically potentiated rapamycin’s suppression of hsBAFF-induced Erk1/2 activation and B-cell proliferation/viability (Fig. 3B–G).

We recently have presented a role for mTORC1/2 downstream of the hsBAFF-induced [Ca²⁺]_i elevations [26; 62]. In this study, interestingly, rapamycin is involved in an upstream role for mTORC1/2 in mediating hsBAFF-induced [Ca²⁺]_i elevations in B cells. This supported by the finding that mTORC1/2 inhibitor PP242 or Akt inhibitor X, like rapamycin, relieved the basal or hsBAFF-induced [Ca²⁺]_i elevations (Fig.4A). Next, we employed BAPTA/AM (an intracellular Ca²⁺ chelator), EGTA (an extracellular Ca²⁺ chelator), 2-APB (an inhibitor for both IP3 receptors and CRAC channels), ML-SA1 (an agonist of TRPML1 channel for lysosomal Ca²⁺ release) or verapamil (a blocker of TRPML1 channel) as pharmacological chelators, activator or inhibitor to pinpoint whether rapamycin inhibits hsBAFF-stimulated B-cell proliferation/viability via the PTEN/Akt-Erk1/2 signaling pathway in a Ca²⁺-dependent manner. We demonstrated that chelating intracellular Ca²⁺ with BAPTA/AM or preventing [Ca²⁺]_i elevation using EGTA, 2-APB or verapamil reinforced rapamycin’s suppression of hsBAFF-stimulated [Ca²⁺]_i elevations, PTEN/Akt-Erk1/2 signaling pathway, and proliferation/viability in B cells (Fig, 4–6). However, elevating [Ca²⁺]_i by ML-SA1 conferred high resistance to rapamycin’s inhibition of hsBAFF-evoked events in the cells (Fig. 6). These findings underline that rapamycin counteracts hsBAFF-stimulated B-cell proliferation and survival by hindering mTORC1/2 and blocking hsBAFF-induced [Ca²⁺]_i elevations, thereby hindering the PTEN/Akt-Erk1/2 signaling pathway. However, there are still some shortcomings in this study. For example, feedback mechanisms of mTORC1/2 on Ca²⁺ channels such as IP3Rs remain to be further studied.

It has been reported that anti-apoptotic Bcl-2-family members are multimodal regulators of intracellular Ca²⁺ signaling events in cell survival and death [63]. Therefore, a question that arises from the current work is whether the remodeling of Bcl-2-family proteins due to hsBAFF and/or rapamycin affects Ca²⁺ homeostasis and dynamics in B cells. We noticed that hsBAFF (2.5 μg/ml) obviously upregulated the expressions of Mcl-1, Bcl-2, Bcl-xL and survivin at 8–12 h with a concomitant [Ca²⁺]_i elevations in Raji cells, Daudi cells and/or purified mouse B lymphocytes (Fig. 1A and 2E), and rapamycin downregulation of the proteins’ increases was in line to rapamycin attenuation of the [Ca²⁺]_i elevations in the cells in response to hsBAFF (Fig.1 and 2). The data clearly indicate that hsBAFF-remodeled anti-apoptotic Bcl-2-family members may accordingly evoke positive feedback for Ca²⁺ signals, thereby promoting B-cell proliferation/survival, and the overall events of which are modulated by rapamycin.

CaMKII has been reported to regulate the development and activity of many different cell types including immune cells through Ca²⁺ signaling [21; 22; 23; 24]. Since CaMKII acts as a general integrator of Ca²⁺ signaling, we speculated that rapamycin likely intervenes in the PTEN/Akt-Erk1/2 signaling pathway and B-cell proliferation/viability by inhibiting Ca²⁺-dependent CaMKII phosphorylation. Indeed, in this study, we showed that rapamycin blocked hsBAFF-elicited phosphorylation of CaMKII (Fig. 7A), and pretreatment with BAPTA/AM, EGTA or 2-APB strengthened rapamycin’s suppression of hsBAFF-increased phosphorylation of CaMKII in Raji cells and purified mouse B lymphocytes (Fig. 7B), suggesting that rapamycin disturbs hsBAFF elevation of [Ca²⁺]_i-mediated CaMKII phosphorylation. To clarify whether CaMKII activity is essential for rapamycin’s prevention of hsBAFF-evoked events in the B cells, KN93, a specific inhibitor of CaMKII [64], was utilized. We found that KN93 potently enhanced the inhibitory effects of rapamycin on the phosphorylation of PTEN, Akt and Erk1/2, as well as the expression of Bcl-2 and survivin in Raji cells and purified mouse B lymphocytes induced by hsBAFF (Fig. 7C), and effectively strengthened rapamycin’s inhibition of B-cell proliferation/viability (Fig. 7D and E). Similar results were also observed in the cells treated with lentiviral shRNA to CaMKII (Fig. 7F–I). These results strongly support that rapamyicn inhibits hsBAFF-stimulated B-cell proliferation and survival by mediating Ca²⁺-CaMKII-dependent PTEN/Akt-Erk1/2 signaling pathway. Our data provide an expanded conceptual view of rapamycin’s intervention in BAFF-stimulated proliferation and survival signals, which should contribute to understanding of the action of rapamycin as an effective agent for prevention of excessive BAFF-induced aggressive or neoplastic B-cell disorders.

In summary, here we have identified that rapamycin attenuation of hsBAFF-elevated [Ca²⁺]_i linked to its inhibition of proliferation and survival in normal and neoplastic B-lymphoid cells. Mechanistically, rapamycin inhibited hsBAFF-stimulated cell proliferation by arresting cells at G₀/G₁ phase, which was related to downregulating the protein levels of CDK2, CDK4, CDK6, cyclin A, cyclin D1, and cyclin E. Rapamycin inhibited hsBAFF-stimulated cell survival by downregulating the levels of anti-apoptotic proteins (Mcl-1, Bcl-2, Bcl-xL and survivin) and meanwhile upregulating the levels of pro-apoptotic proteins (BAK and BAX). We demonstrated that rapamyicn inhibited hsBAFF-stimulated B-cell proliferation and survival by blunting mTORC1/2-mediated [Ca²⁺]_i elevations and suppressing Ca²⁺-CaMKII-dependent PTEN/Akt-Erk1/2 signaling pathway (Fig. 8). Our finding underscores that rapamycin may be exploited for the prevention of excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.

Fig. 8.

A schematic model of how rapamycin inhibits hsBAFF-stimulated B-cell proliferation and survival by Ca²⁺ signaling. Rapamycin prevents B cells from hsBAFF-stimulated proliferation/survival by blunting mTORC1/2-mediated [Ca²⁺]_i elevations and suppressing Ca²⁺-CaMKII-dependent PTEN/Akt-Erk1/2 signaling pathway.

Highlights.

• Rapamycin attenuation of BAFF-elevated [Ca²⁺]_i links to its inhibition of B-cell proliferation/survival.

• Rapamyicn blunts mTORC1/2-mediated [Ca²⁺]_i elevations induced by BAFF.

• Rapamyicn inhibits BAFF-stimulated B-cell proliferation/survival by blocking Ca²⁺-CaMKII-dependent PTEN/Akt-Erk1/2 signaling.

• Rapamycin may be an effective agent for prevention of BAFF-induced aggressive or neoplastic B-cell disorders.

Abbreviations:

2-APB

2-aminoethoxydiphenyl borate

4E-BP1

eukaryotic initiation factor 4E binding protein 1

Akt

protein kinase B (PKB)

BAFF

B-cell activating factor of the TNF family

BAPTA/AM

1,2-bis(o-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid tetra(acetoxymethyl) ester

BLyS

B lymphocyte stimulator

BCMA

B cell maturation antigen

Ca²⁺

calcium ion

CaM

calmodulin

CaMKII

calcium/calmodulin-dependent protein kinase II

CDK

cyclin-dependent kinase

CRAC

Ca²⁺-release activated Ca²⁺

EGTA

ethylene glycol tetra-acetic acid

Erk1/2

extracellular signal-related kinases 1/2

FBS

fetal bovine serum

GFP

green fluorescent protein

MAPK

mitogen-activated protein kinase

MKK

mitogen-activated protein kinase kinase

mTOR

mammalian target of rapamycin

mTORC1/2

mTOR complexes 1/2

PTEN

phosphatase and tensin homologue deleted on chromosome 10

PBS

phosphate buffered saline

PI3K

phosphatidylinositol 3′-kinase

RA

rheumatoid arthritis

S6K1

ribosomal protein S6 kinase 1

SLE

systemic lupus erythematosus

SS

Sjögren’s syndrome

TACI

transmembrane activator and cyclophilin ligand interactor

TALL-1

TNF and apoptosis ligand-related leukocyte-expressed ligand 1

THANK

TNF homologue that activates apoptosis, nuclear factor κB, and c-Jun NH2-terminal kinase

TRPML1

transient receptor potential mucolipin 1