Rapamycin inhibits BAFF-stimulated cell proliferation and survival by suppressing mTOR-mediated PP2A-Erk1/2 signaling pathway in normal and neoplastic B-lymphoid cells

Abstract

B-cell activating factor (BAFF) is involved in not only physiology of normal B cells, but also pathophysiology of aggressive B cells related to malignant and autoimmune diseases. Rapamycin, a lipophilic macrolide antibiotic, has recently shown to be effective in the treatment of human lupus erythematosus. However, how rapamycin inhibits BAFF-stimulated B-cell proliferation and survival has not been fully elucidated. Here, we show that rapamycin inhibited human soluble BAFF (hsBAFF)-induced cell proliferation and survival in normal and B-lymphoid (Raji and Daudi) cells by activation of PP2A and inactivation of Erk1/2. Pretreatment with PD98059, down-regulation of Erk1/2, expression of dominant negative MKK1, or overexpression of wild-type PP2A potentiated rapamycin’s suppression of hsBAFF-activated Erk1/2 and B-cell proliferation/viability, whereas expression of constitutively active MKK1, inhibition of PP2A by okadaic acid, or expression of dominant negative PP2A attenuated the inhibitory effects of rapamycin. Furthermore, expression of a rapamycin-resistant and kinase-active mTOR (mTOR-T), but not a rapamycin-resistant and kinase-dead mTOR-T (mTOR-TE), conferred resistance to rapamycin’s effects on PP2A, Erk1/2 and B-cell proliferation/viability, implying mTOR-dependent mechanism involved. The findings indicate that rapamycin inhibits BAFF-stimulated cell proliferation/survival by targeting mTOR-mediated PP2A-Erk1/2 signaling pathway in normal and neoplastic B-lymphoid cells. Our data highlight that rapamycin may be exploited for preventing excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.

Introduction

The B-cell activating factor from the TNF family (BAFF), also known as BLyS, TALL-1, THANK, and zTNF4, a type II membrane protein that exists in both membrane-bound and soluble forms, is known to be crucial for development, maturation and homeostasis of normal B lymphocytes [1–4]. It exerts tonic effects on B-cell proliferation and survival via three receptors: BAFF-R (BR3), BCMA and TACI [5–7]. BAFF plays a critical role in normal B lymphocyte development, but excessive expression of BAFF causes various autoimmune diseases [8, 9]. Multiple studies have demonstrated that BAFF is the culprit of malignant B-cell proliferation [8, 10, 11]. Excessive endogenous and transgenic BAFF in mice prolong the life span and increase the population of peripheral B lymphocytes, which results in elevated secretion of superfluous autoantibodies [8, 12]. In human, increased levels of serum BAFF are involved in a number of autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjögren’s syndrome (SS) [8, 13, 14]. Elegant experiments have shown the etiological mechanism of excess BAFF in aggressive B-cell proliferation and survival [15]. Hence, high levels of BAFF, which contribute to aggressive or neoplastic B-cell disorders, have been thought to actually play a central role in the pathophysiology of autoimmune diseases [16, 17].

Mounting studies have demonstrated that BAFF regulates expression of several Bcl-2 family members, including Bcl-xL, Mcl-1, A1/Bfl-1, Bcl-2, and Bim, via survival-promoting kinase systems such as Pim 1/2 or extracellular signal-related kinases 1/2 (Erk1/2) [7]. BAFF also activates Akt and mammalian target of rapamycin (mTOR) pathways, promoting B-cell growth and survival [18, 19]. Rapamycin, a lipophilic macrolide antibiotic that regulates mitochondrial transmembrane potential and Ca²⁺ fluxing in T cells, has been successfully used in renal transplantation [20]. In fact, rapamycin is a potent and allosteric inhibitor of mTOR [21, 22]. Of interest, it has recently been shown that rapamycin is effective in the treatment of human SLE, by decreasing the basal calcium level and T cell receptor-induced calcium influx [23]. Furthermore, we have observed that pretreatment with rapamycin potently inhibited human soluble BAFF (hsBAFF)-stimulated B-cell proliferation and survival by preventing elevation of intracellular free Ca²⁺ ([Ca²⁺]_i) and phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) [24]. In addition, we have found that the inhibitory effects of rapamycin on B-cell proliferation and survival was associated with the blockage of hsBAFF-stimulated phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1) [24], two best characterized downstream effector molecules of mTOR [21, 22]. However, it is not clear whether the inhibitory activity is also attributed to rapamycin’s inhibition of mTOR-mediated other signaling pathways responsible for B-cell proliferation and survival.

PP2A, a ubiquitous and highly conserved serine/threonine (Ser/Thr) protein phosphatase, is responsible for controlling numerous cellular processes, including cell growth/proliferation, cell survival/death, and cell mobility, as well as multiple signaling pathways [25–27]. Dysregulation of PP2A activity has been reported in several autoimmune diseases. For example, there exists an increased PP2A activity in T cells from patients with SLE [28]. Overexpression of PP2A evokes DNA hypomethylation through suppressing MKK/Erk/DNA methyltransferase 1 (DNMT1) pathway in normal and SLE T cells [27]. However, decreased PP2A activity promotes IL-23 production contributing to excessive numbers of Th17 cells in experimental autoimmune encephalomyelitis (EAE) [29]. Accumulative data have pointed to an important role of PP2A in developmental processes of autoimmune disease such as SLE [27, 30, 31].

PP2A, as a negative regulator, dephosphorylates and inactivates both mitogen-activated protein kinase kinases 1/2 (MKK1/2) and Erk1/2 [25]. The activation of Erk1/2 in B lymphocytes is dependent on calcium flux [32]. Our previous studies have shown that hsBAFF-induced [Ca²⁺]_i elevation activates Erk1/2 pathway contributing to proliferation and survival of primary mouse B lymphocytes [33]. We have also pinpointed that excessive hsBAFF activates Erk1/2 promoting cell proliferation and survival by inhibition of PP2A in normal and neoplastic B-lymphoid cells [34]. It has been described that mTOR mediates phosphorylation of S6K1 and 4E-BP1 by inhibiting PP2A [35]. Besides, rapamycin can activate PP2A [35]. Therefore, we were interested in determining whether rapamycin’s suppression of BAFF-stimulated B-cell proliferation and survival is also associated with its inhibition of mTOR-mediated PP2A-Erk1/2 signaling pathway.

Here, we show that rapamycin inhibits hsBAFF-stimulated cell proliferation and survival by activation of PP2A, thereby resulting in inhibition of Erk1/2 pathway in normal and neoplastic B-lymphoid cells. Furthermore, we demonstrate that the effect of rapamycin on PP2A-Erk1/2 signaling pathway is through inhibition of mTOR in the cells. Our data highlight that rapamycin may be exploited for prevention of excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.

Materials and methods

Reagents

Anti-CD19 magnetic fluorobeads-B was purchased from One Lambda (Canoga Park, CA, USA). Okadaic acid and PD98059 were purchased from Sigma (St. Louis, MO, USA). RPMI 1640 Medium was from Gibco (Rockville, MD, USA). Fetal bovine serum (FBS) was supplied by Hyclone (Logan, UT, USA). Refolded human soluble BAFF (hsBAFF) was a recombinant form of the extracellular domain of the BAFF synthesized in Escherichia coli from this group [36]. CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay kit was from Promega (Madison, WI, USA). Annexin-V-FITC/Propidium Iodide (PI) Apoptosis Detection kit was obtained from BD Biosciences (San Diego, CA, USA). Enhanced chemiluminescence solution was from Millipore (Billerica, MA, USA). The following antibodies were used: PP2ACα (BD Biosciences, San Jose, CA, USA), PP2A-A subunit, PP2A-B subunit (Millipore, Billerica, MA, USA), 4E-BP1, phospho-4E-BP1 (Thr70), phospho-Erk1/2 (Thr202/Tyr204), phospho-S6K1 (Thr389) (Cell Signaling Technology, Beverly, MA, USA), β-actin, Erk2, demethylated-PP2A, S6K1, CDK2, CDK4, CDK6, cyclin A, cyclin D1, cyclin E, Cdc25A, Cdc25B, p21, p27 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-PP2A (Epitomics, Burlingame, CA, USA), FLAG, HA, MKK1, mTOR (Sigma), goat anti-rabbit IgG-horseradish peroxidase (HRP), goat anti-mouse IgG-HRP, and rabbit anti-goat IgG-HRP (Pierce, Rockford, IL, USA). Other chemicals were purchased from local commercial sources and were of analytical grade.

Cells

Raji and Daudi cell lines were from American Type Culture Collection (ATCC) (Manassas, VA, USA). Cells 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. Normal mouse B lymphocytes were purified from fresh splenic cells of healthy mice using anti-CD19 magnetic fluorobeads and cultured as described previously [24].

Recombinant adenoviral constructs and infection of cells

The recombinant adenoviruses expressing FLAG-tagged rapamycin-resistant and kinase-active mTOR (S2035T, designated Ad-mTOR-T), FLAG-tagged rapamycin-resistant and kinase-dead mTOR-T (S2035T/D2357E, designated Ad-mTOR-TE), FLAG-tagged wild-type rat PP2ACα (Ad-PP2A), FLAG-tagged constitutively active MKK1 (Ad-MKK1-R4F), FLAG-tagged dominant negative MKK1 (Ad-MKK1-K97M), hemagglutinin (HA)-tagged dominant negative (dn) PP2A catalytic subunit (PP2Ac) (L199P) (dn-PP2A), and the control vector expressing green fluorescent protein (GFP) alone (Ad-GFP) were described previously [37–39]. For experiments, 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, cells were used for experiments. Ad-GFP served as a control. Expression of FLAG-tagged mTOR, PP2A, MKK1 and HA-tagged dn-PP2A was determined by determined by Western blot analysis with antibodies to FLAG and HA, respectively.

Lentiviral shRNA cloning, production and infection

Lentiviral shRNAs to mTOR, Erk1/2 and GFP (for control) were generated and used as described [40].

Cell proliferation and viability assay

Purified mouse B lymphocytes, Raji and/or Daudi cells, or Raji cells infected with Ad-mTOR-T, Ad-mTOR-TE, Ad-PP2A, Ad-dn-PP2A, Ad-MKK1-R4F, Ad-MKK1-K97M and Ad-GFP, respectively, or Raji cells infected with lentiviral shRNAs to Erk1/2 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) under standard culture conditions and kept overnight at 37 °C humidified incubator with 5 % CO₂. The next day, cells were treated with/without hsBAFF (2.5 μg/ml) for 48 h following pre-incubation with/without rapamycin (50–200 or 100 ng/ml) for 2 h, or with/without okadaic acid (100 nM) or PD98059 (10 μM) for 1 h and then hsBAFF (2.5 μg/ml) for 48 h following pre-incubation with/without rapamycin (100 ng/ml) for 2 h with 3–6 replicates of each treatment. Subsequently, cell proliferation was assessed by counting the trypsinized cells with a Coulter Counter (Beckman Coulter, Fullerton, CA, USA). The viability of the cells, after incubation with MTS reagent (one solution reagent) (20 μl/well) for 4 h, was determined by measuring the optical density (OD) at 490 nm using a Synergy™ 2 Multi-function Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA).

Live cell detection by trypan blue exclusion and flow cytometry

Purified mouse B lymphocytes, Raji and Daudi cells were seeded in 24-well plates (3 × 10⁵ cells/well, for trypan blue exclusion) or 6-well plates (2 × 10⁶ cells/well, for flow cytometry), respectively. The next day, cells were treated with/without hsBAFF (2.5 μg/ml) for 48 h following pre-incubation with/without rapamycin (50–200 ng/ml) for 2 h. Then, live cells were recorded by counting viable cells using trypan blue exclusion, and the ratios of dead cells, live cells, necrotic and apoptotic cells were monitored by a fluorescence-activated cell sorter (FACS) Vantage SE flow cytometer (Becton Dickinson, CA, USA) using annexin-V-FITC/PI staining.

In vitro PP2A phosphatase assay

Purified mouse B lymphocytes, Raji and Daudi cells were lysed in 50 mM Tris–HCl buffer, pH 7.0, containing 1 % Nonidet P-40, 2 mM EDTA, and protease inhibitor cocktail (Sigma, 1:1000). PP2Ac was immunoprecipitated with antibodies to PP2Ac (Millipore, Temecula, CA, USA), and protein A/G agarose (Santa Cruz Biotechnology). Subsequently, the beads were washed three times with the above lysis buffer, and twice with the phosphatase assay buffer (50 mM Tris–HCl, pH 7.0, 0.1 mM CaCl₂). The phosphatase activity of immunoprecipitated PP2A was assayed with a Ser/Thr Phosphatase Assay kit 1 using KRpTIRR as the substrate peptide (Millipore) following the manufacturer’s instructions. PP2A activity was detected by measuring OD values at 595 nm using a SynergyTM 2 Multi-function Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA). Because OD values varied from experiment to experiment, PP2A activity was calculated using the percentage change in each experiment. Finally, all data (from different batches of experiments) were pooled for statistical analysis.

Western blot analysis

Purified mouse B lymphocytes, Raji cells, or Raji cells infected with Ad-mTOR-T, Ad-mTOR-TE, Ad-PP2A, Ad-dn-PP2A, Ad-MKK1-R4F, Ad-MKK1-K97M and Ad-GFP, respectively, or Raji cells infected with lentiviral shRNAs to Erk1/2 and GFP, respectively, 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, cells were treated with/without 2.5 μg/ml hsBAFF for 12 h following pre-incubation with/without rapamycin (50–200 or 100 ng/ml) for 2 h or PD98059 (0.1–25 μM) for 1 h, or with/without okadaic acid (100 nM) or PD98059 (1 or 10 μM) for 1 h and then hsBAFF (2.5 μg/ml) for 12 h following pre-incubation with/without rapamycin (50 or 100 ng/ml) for 2 h. Afterwards, Western blotting was performed as described previously [40].

Statistical analysis

All quantified data were expressed as mean values ± standard error (mean ± SE). The Student’s t test for non-paired replicates was used to identify statistically significant differences between treatment means. Group variability and interaction were compared using either one-way or two-way ANOVA followed by Bonferroni’s post-tests to compare replicate means. Significance was accepted at P < 0.05.

Results

Rapamycin inhibits hsBAFF-stimulated cell proliferation and survival in normal and lymphoma B cells

We have recently shown that excessive hsBAFF induces B-cell proliferation and survival in part through activation of mTOR pathway, and inhibition of mTOR by rapamycin prevents the effects of hsBAFF on primary mouse B lymphocytes [24]. In line with the above findings, here we also observed that pretreatment of Raji cells, Daudi cells and primary B lymphocytes with rapamycin (50–200 ng/ml) for 2 h reduced hsBAFF-stimulated cell proliferation and viability in a concentration-dependent manner, as evaluated by cell counting (Fig. 1a) and MTS assay (Fig. 1b), respectively.

Fig. 1.

Rapamycin attenuates hsBAFF-induced normal and Raji and Daudi B-cell proliferation and viability. Raji cells, Daudi cells and purified mouse splenic B lymphocytes were pretreated with rapamycin (0–200 ng/ml) for 2 h, and then stimulated with 2.5 μg/ml hsBAFF for 48 h. a Cell proliferation was evaluated by cell counting. b Cell viability was detected by MTS assay. c The relative number of live cells was estimated by trypan blue exclusion assay. d The percentages of live (Q3), early apoptotic (Q4), late apoptotic (Q2) and necrotic cells (Q1) were determined by FACS using annexin-V-FITC/PI staining. The results from a representative experiment are shown. e, f Quantitative analysis of live cells and apoptotic cells by FACS assay. Results are presented as mean ± SE (n = 3–6). ^a P < 0.05, difference vs control group; ^b P < 0.05, difference vs 2.5 µg/ml hsBAFF group

To confirm the event that rapamycin does suppress hsBAFF-induced cell survival, we counted viable cells using trypan blue exclusion (Fig. 1c). Furthermore, we evaluated rapamycin-induced apoptosis by fluorescence-activated cell sorting (FACS) using annexin-V-FITC/PI staining (Fig. 1d–f). As expected, pretreatment with rapamycin significantly decreased the relative number of live cells (Fig. 1c–e) and concurrently increased the relative number of apoptotic cells (Fig. 1d, f) in hsBAFF-stimulated Raji cells, Daudi cells and primary B lymphocytes in a concentration-dependent manner (Fig. 1c–f). Taken together, our results strongly support the notion that rapamycin is a potent agent for intervention in excess BAFF-induced aggressive B-cell proliferation and survival. As 100 ng/ml of rapamycin was able to inhibit the cell proliferation/viability nearly to the basal level, this concentration was selected for further studies, as described below.

Rapamycin intervenes in hsBAFF-induced inhibition of PP2A and activation of Erk1/2 in B cells

Protein phosphatases 2A functions as a key regulator of many cellular events, such as cell proliferation, migration, and survival [25–27, 41]. It has been shown that rapamycin inhibits mTOR-mediated phosphorylation of S6K1 and 4E-BP1 by activation of PP2A [35]. PP2A negatively regulates Erk1/2 pathway through dephosphorylation of Erk1/2 [25]. Our recent studies have demonstrated that excess BAFF inhibits PP2A activity, thereby activating Erk1/2 signaling and promoting B-cell proliferation and survival [34]. Therefore, here we tested whether rapamycin exerts the inhibitory effects on hsBAFF-induced B-cell proliferation/viability through intervening in PP2A-Erk1/2 signaling pathway. To this end, Raji cells, Daudi cells and purified mouse splenic B lymphocytes were pretreated with/without 50–200 ng/ml of rapamycin for 2 h and then stimulated with/without 2.5 μg/ml of hsBAFF for 12 h. As shown in Fig. 2a, hsBAFF-induced expression of demethylated-PP2Ac, phospho-PP2Ac and phospho-Erk1/2 was diminished by 2 h-pretreatment with rapamycin concentration-dependently. At 100 ng/ml, rapamycin was able to suppress hsBAFF-induced demethylated-PP2Ac, phospho-PP2Ac and phospho-Erk1/2 nearly to the basal levels (Fig. 2a), in line with rapamycin’s inhibitory effects on hsBAFF-induced B-cell proliferation/viability (Fig. 1), indicating rapamycin’s activation of PP2A. This is further supported by the finding that rapamycin prevented hsBAFF from inhibiting PP2A activity (Fig. 2b), as determined by the in vitro Ser/Thr phosphatase assay.

Fig. 2.

Rapamycin intervenes in hsBAFF-induced inhibition of PP2A and activation of Erk1/2 in B cells. Raji cells, Daudi cells and purified mouse splenic B lymphocytes were pretreated with rapamycin (0–200 ng/ml) for 2 h, and then stimulated with 2.5 μg/ml hsBAFF for 12 h. a Total cell lysates were subjected to Western blotting using indicated antibodies, showing that rapamycin and/or hsBAFF did not alter cellular protein levels of PP2A-A, PP2A-B, PP2Ac and Erk2, but hsBAFF-induced robust expression of demethylated-PP2A (de-PP2A), phospho-PP2A (p-PP2A), and phospho-Erk1/2 (p-Erk1/2) was attenuated by rapamycin concentration-dependently. The blots were probed for β-actin as a loading control. Similar results were observed in three independent experiments. b PP2A in cell lysates was immunoprecipitated with antibodies to PP2Ac plus protein A/G agarose beads, followed by in vitro phosphatase assay using Ser/Thr Phosphatase Assay Kit 1 (Millipore). Results are presented as mean ± SE (n = 3). ^a P < 0.05, difference vs control group; ^b P < 0.05, difference vs 2.5 µg/ml hsBAFF group

PP2A is a heterotrimeric holoenzyme, which consists of a catalytic subunit (PP2Ac), an A subunit (also termed PR65), and members of the B subunit families, such as B (PR55), B′ (PR61), B″ (PR72), and B′″ (PR93/PR110) [42]. As the localization and the substrate specificity of PP2Ac are regulated by its association with PP2A-A and -B regulatory subunits [42], we also tested whether rapamycin and/or hsBAFF influences expression of PP2A-A or PP2A-B. It turned out that hsBAFF and/or rapamycin did not alter cellular protein levels of PP2A-A or PP2A-B (Fig. 2a). Collectively, the results imply that rapamycin inhibition of excess hsBAFF-induced B-cell proliferation and survival is associated with rapamycin’s intervention in hsBAFF-induced inactivation of PP2A and activation of Erk1/2.

Rapamycin inhibits hsBAFF-induced B-cell proliferation and viability by blocking Erk1/2 pathway

To substantiate whether rapamycin inhibition of hsBAFF-induced cell proliferation/viability is related to rapamycin blockage of Erk1/2 activation, Raji cells and purified mouse splenic B lymphocytes were pre-incubated with/without PD98059 (a selective inhibitor of MKK1/2, upstream of Erk1/2) alone, or in combination with rapamycin. We found that PD98059 (0.1–25 µM) concentration-dependently inhibited hsBAFF-induced phosphorylation of Erk1/2 in the cells (Fig. 3a). PD98059 (1 µM) or rapamycin (50 ng/ml) alone slightly suppressed the basal or hsBAFF-induced phosphorylation of Erk1/2. However, co-treatment with 50 g/ml of rapamycin and 1 μM of PD98059, like the treatment with 100 ng/ml of rapamycin or 10 µM of PD98059 alone, exhibited an obvious inhibitory effect on phospho-Erk1/2 in the cells (Fig. 3b). Of interest, treatment with 50 ng/ml of rapamycin or 1 μM of PD98059 alone inhibited cell proliferation and cell viability slightly, whereas co-treatment with rapamycin (50 ng/ml)/PD98059 (1 µM) inhibited the basal or hsBAFF-stimulated B-cell proliferation/viability more potently than treatment with rapamycin or PD98059 alone (Fig. 3c, d). Furthermore, we also showed that the inhibitory effect of co-treatment with rapamycin (50 ng/ml)/PD98059 (1 µM) was in line with that of treatment with 100 ng/ml of rapamycin or 10 µM of PD98059 alone, but significantly weaker than that of co-treatment with rapamycin (100 ng/ml)/PD98059 (10 µM) (Fig. 3c, d). Similarly, U0126 (0.5 μM), another MKK1/2 inhibitor, was able to potentiate the inhibitory effects of rapamycin as well (data not shown).

Fig. 3.

Pharmacological inhibition of Erk1/2 with PD98059 enhances rapamycin’s prevention of hsBAFF-induced cell proliferation/viability in B cells. Raji cells and purified mouse splenic B lymphocytes were treated with/without 2.5 μg/ml hsBAFF for 12 h (for Western blotting) following pre-incubation with/without PD98059 (0.1–25 μM) for 1 h, or with/without PD98059 (1 or 10 μM) for 1 h and then hsBAFF (2.5 μg/ml) for 12 h (for Western blotting) or 48 h (for cell proliferation/viability assay) following pre-incubation with/without rapamycin (50 or 100 ng/ml) for 2 h. Total cell lysates were subjected to Western blotting using indicated antibodies (a, b). The blots were probed for β-actin as a loading control. Similar results were observed in at least three independent experiments. The cell proliferation was evaluated by cell counting (c) and the cell viability was determined by the MTS assay (d). a, b PD98059 inhibited hsBAFF-induced phosphorylation of Erk1/2 concentration-dependently (a), and PD98059 (1 µM) or rapamycin (50 ng/ml) alone slightly suppressed the basal or hsBAFF-induced phosphorylation of Erk1/2, whereas co-treatment with 50 g/ml of rapamycin and 1 μM of PD98059, like the treatment with 100 ng/ml of rapamycin or 10 µM of PD98059 alone, exhibited an obvious inhibitory effect on phospho-Erk1/2 in the cells (b). c, d Treatment with 50 ng/ml of rapamycin or 1 μM of PD98059 alone showed a slight inhibition of cell proliferation (c) and cell viability (d), whereas co-treatment with rapamycin (50 ng/ml)/PD98059 (1 µM) exhibited more potent inhibition of the basal or hsBAFF-stimulated B-cell proliferation/viability than rapamycin or PD98059 alone. The inhibitory effect of co-treatment with rapamycin (50 ng/ml)/PD98059 (1 µM) was in line with that of treatment with 100 ng/ml of rapamycin or 10 µM of PD98059 alone, but significantly weaker than that of co-treatment with rapamycin (100 ng/ml)/PD98059 (10 µM). Results are presented as mean ± SE (n = 6). ^a P < 0.05, difference vs control group; ^b P < 0.05, difference vs 2.5 µg/ml hsBAFF group; ^c P < 0.05, difference vs hsBAFF/rapamycin group or hsBAFF/PD98059 group

To further investigate the role of Erk1/2 in rapamycin’s inhibition of hsBAFF-induced B-cell proliferation/viability, we carried out gene silencing or gene overexpression experiments. At first, expression of Erk1/2 was silenced by RNA interference. As shown in Fig. 4a, lentiviral shRNA to Erk1/2, but not to GFP, down-regulated the expression of Erk1/2 protein by ~90 % in Raji cells. Silencing Erk1/2 markedly attenuated the basal or hsBAFF-induced phosphorylation of Erk1/2, as detected by Western blotting (Fig. 4b). Consistently, down-regulation of Erk1/2 conferred significant reduction of the basal or hsBAFF-stimulated proliferation/viability in Raji cells as well (Fig. 4c, d). Importantly, addition of rapamycin exhibited more inhibitory effects on hsBAFF-induced phosphorylation of Erk1/2 and proliferation/viability in the cells infected with lentiviral shRNA to Erk1/2 than in the cells infected with lentiviral shRNA to GFP (Fig. 4b–d).

Fig. 4.

Down-regulation of Erk1/2, expression of constitutively active or dominant negative MKK1 influences rapamycin’s inhibition of hsBAFF-induced cell proliferation/viability in B cells. Raji cells, infected with lentiviral shRNA to Erk/2 or GFP (as control), or with Ad-MKK1-R4F, Ad-MKK1-K97M and Ad-GFP (as control), respectively, were treated with/without 2.5 μg/ml hsBAFF for 12 h (for Western blotting) or 48 h (for cell proliferation/viability assay) following pre-incubation with/without rapamycin (100 ng/ml) for 2 h. Total cell lysates were subjected to Western blotting using indicated antibodies (a, b, e, h). The blots were probed for β-actin as a loading control. Similar results were observed in at least three independent experiments. The cell proliferation was evaluated by cell counting (c, f) and the cell viability was determined by the MTS assay (d, g). a Lentiviral shRNA to Erk/2, but not to GFP, down-regulated Erk1/2 expression by ~90 % in Raji cells. b Silencing Erk1/2 obviously potentiated rapamycin’s inhibition of hsBAFF-induced phosphorylation of Erk1/2. c, d Down-regulation of Erk1/2 conferred significant reduction of the basal or hsBAFF-stimulated proliferation/viability in Raji cells and enhanced rapamycin’s inhibitory effects. e Infection of Raji cells with Ad-MKK1-R4F and Ad-MKK1-K97M, but not Ad-GFP, resulted in expression of high levels of FLAG-tagged MKK1 mutants. Expression of MKK1-R4F resulted in robust phosphorylation of Erk1/2 even without stimulation with hsBAFF, whereas expression of MKK1-K97M suppressed hsBAFF-stimulated phosphorylation of Erk1/2. f, g Expression of MKK1-R4F markedly elevated the basal or hsBAFF-stimulated cell proliferation/viability, and conferred profound resistance to rapamycin’s inhibitory effects. In contrast, expression of MKK1-K97M potently inhibited the events and reinforced rapamycin’s action. h Silencing Erk1/2 remarkably inhibited the expression of cyclin A, cyclin E, cyclin D1, CDK2, CDK4 and CDK6, concurrently strongly upregulated p21 and p27 in Raji cells stimulated with or without hsBAFF. Silencing Erk1/2 strengthened the inhibitory effect of rapamycin on hsBAFF-induced expression of the above proteins, but failed to further increase the expression of p21 and p27 in the presence of rapamycin. Results are presented as mean ± SE (n = 6). ^a P < 0.05 difference vs control group; ^b P < 0.05, difference vs 2.5 µg/ml hsBAFF group; ^c P < 0.05 Erk1/2 shRNA group vs GFP shRNA group; ^d P < 0.05, Ad-MKK1-R4F group or Ad-MKK1-K97M group vs Ad-GFP group

Next, we employed recombinant adenoviruses Ad-MKK1-R4F and Ad-MKK1-K97M, encoding FLAG-tagged constitutively active and dominant negative MKK1, respectively. Infection of Raji cells with Ad-MKK1-R4F and Ad-MKK1-K97M, but not Ad-GFP (control virus), resulted in expression of high levels of FLAG-tagged MKK1 mutants (Fig. 4e). Expression of MKK1-R4F led to robust phosphorylation of Erk1/2 even without stimulation with hsBAFF, whereas expression of MKK1-K97M blocked hsBAFF-stimulated phosphorylation of Erk1/2 (Fig. 4e), indicating that the MKK1 mutants function in the cells as expected. Of note, expression of MKK1-R4F in Raji cells significantly elevated the basal or hsBAFF-stimulated cell proliferation/viability, and conferred profound resistance to rapamycin’s inhibitory effects (Fig. 4f, g). In contrast, expression of MKK1-K97M in the cells dramatically reduced the basal or hsBAFF-stimulated cell proliferation/viability, and especially addition of rapamycin reinforced the events (Fig. 4f, g). The results clearly indicate that rapamycin inhibits hsBAFF-stimulated cell proliferation/viability, in part by blocking Erk1/2 pathway in B cells.

It is known that cell proliferation is associated with cell cycle progression [43]. Rapamycin inhibits proliferation/growth of malignant B cells by affecting expression of key regulatory proteins related to the G1/S cell cycle progression [44, 45]. The activated Erk1/2 stimulates the transcription of the cyclin-dependent kinases (CDK) CDK4, CDK6 and cyclin D genes [46]. Pharmacological inhibition of Erk1/2 potently decreases CDK2, CDK4, cyclin D1 and cyclin E expression, and increases high levels of CDK inhibitors p21 and p27 [47, 48]. Therefore, we examined the expression of G1-CDKs and related regulatory proteins, including cyclins, Cdc25 and CDK inhibitors in Raji cells infected with lentiviral shRNA to Erk1/2 or GFP. As shown in Fig. 4h, the expression of cyclin A, cyclin E, cyclin D1, CDK2, CDK4 and CDK6 was remarkably inhibited, while the expression of two CDK inhibitors p21 and p27 was strongly upregulated in Erk1/2-silenced Raji cells stimulated with or without hsBAFF. Rapamycin was able to markedly block the basal or hsBAFF-stimulated expression of cyclin A, cyclin E, cyclin D1, CDK2, CDK4 and CDK6 in the cells, regardless of infection with lentiviral shRNA to Erk1/2 or not (Fig. 4h). Silencing Erk1/2 enhanced the inhibitory effect of rapamycin on hsBAFF-stimulated expression of the above proteins, but failed to further increase the expression of p21 and p27 in the presence of rapamycin (Fig. 4h). Protein levels of other molecules including Cdc25A and Cdc25B were not obviously altered in the cells (Fig. 4h). Collectively, the findings support the notion that rapamycin’s inhibition of Erk1/2 pathway results in cell proliferation inhibition, at least in part by down-regulating the expression of cyclin A, cyclin E, cyclin D1, CDK2, CDK4 and CDK6, as well as by up-regulating the expression of CDK inhibitors p21 and p27.

Rapamycin blocks hsBAFF-induced Erk1/2 activation and proliferation/viability via PP2A-dependent mechanism in B cells

To pinpoint the role and significance of PP2A in rapamycin’s inhibition of hsBAFF-stimulated B-cell proliferation and survival, we used okadaic acid, a relatively specific PP2A inhibitor [49]. Because okadaic acid, at concentrations up to 100 nM, functions only as a selective inhibitor of PP2A without inhibiting PP1 in intact cells [49], Raji cells and purified mouse splenic B lymphocytes were pretreated with/without rapamycin (100 ng/ml) for 2 h, and then with/without okadaic acid (100 nM) for 1 h, followed by exposure to hsBAFF (2.5 μg/ml) for 12 or 48 h. As shown in Fig. 5a, hsBAFF markedly stimulated the expression of demethylated-PP2Ac, phospho-PP2Ac and phospho-Erk1/2 in the absence or presence of okadaic acid. The effects appeared more potent in the cells co-treated with hsBAFF/okadaic acid than in those treated with hsBAFF or okadaic acid alone (Fig. 5a). Okadaic acid alone profoundly increased the basal levels of demethylated-PP2Ac, phospho-PP2Ac and phospho-Erk1/2 in the absence of rapamycin, and especially reversed the inhibitory effect of rapamycin on haBAFF-stimulated events (Fig. 5a). Next, we investigated whether rapamycin’s inhibition of hsBAFF-stimulated B-cell proliferation and survival is due to the activation of PP2A. Consistent with our previous findings [24], rapamycin inhibited the basal or hsBAFF-stimulated cell proliferation/viability in Raji cells and primary B lymphocytes (Fig. 5b, c). hsBAFF stimulated the cell proliferation/viability, which was dramatically attenuated by rapamycin to or below the basal levels (Fig. 5b, c). Okadaic acid alone obviously strengthened hsBAFF-stimulated cell proliferation/viability although it did not significantly affect the basal value (Fig. 5b, c). Of note, okadaic acid conferred high resistance to rapamycin’s inhibition of hsBAFF-stimulated proliferation/viability in the cells (Fig. 5b, c). These results imply that rapamycin inhibits hsBAFF-induced B-cell proliferation and survival not only via mTOR, but also through PP2A pathway.

Fig. 5.

Pharmacological inhibition of PP2A with okadaic acid confers resistance to rapamycin’s inhibition of hsBAFF-induced activation of Erk1/2 and proliferation/viability in B cells. Raji cells and purified mouse splenic B lymphocytes were treated with/without okadaic acid (100 nM) for 1 h and then hsBAFF (2.5 μg/ml) for 12 h (for Western blotting) or 48 h (for cell proliferation/viability assay) following pre-incubation with/without rapamycin (100 ng/ml) for 2 h. Total cell lysates were subjected to Western blotting using indicated antibodies (a). The blots were probed for β-actin as a loading control. Similar results were observed in at least three independent experiments. The cell proliferation was evaluated by cell counting (b) and the cell viability was determined by the MTS assay (c). a Okadaic acid conferred resistance to rapamycin’s inhibition of hsBAFF-increased de-PP2A, p-PP2A, and p-Erk1/2. b, c Okadaic acid conferred high resistance to rapamycin’s inhibition of hsBAFF-induced cell proliferation/viability. Results are presented as mean ± SE (n = 6). ^a P < 0.05, difference vs control group; ^b P < 0.05, difference vs 2.5 µg/ml hsBAFF group; ^c P < 0.05, difference vs hsBAFF/rapamycin group or hsBAFF/okadaic acid group

To gain more insights into the event that the activity of PP2A is responsible for the inhibitory effects of rapamycin on hsBAFF-induced Erk1/2 activation and proliferation/viability, Raji cells, infected with Ad-dn-PP2A, Ad-PP2A and Ad-GFP (as control), respectively, were pretreated with/without rapamycin (100 ng/ml) for 2 h, and then with/without PD98059 (10 μM) for 1 h, followed by treatment with hsBAFF (2.5 μg/ml) for 12 or 48 h. A low basal level of phospho-Erk1/2 was observed in Ad-GFP-infected cells (control), whereas a higher basal level of phospho-Erk1/2 could be detected in Ad-dn-PP2A-infected cells (Fig. 6a), suggesting the dn-PP2A was functioning in the cells. hsBAFF was able to stimulate the phosphorylation of Erk1/2 in Raji/Ad-GFP and Raji/Ad-dn-PP2A cells (Fig. 6a). Also, due to failure to activate PP2A in the cells expressing dn-PP2A, rapamycin did not significantly inhibit hsBAFF-induced phospho-Erk1/2 in Raji/Ad-dn-PP2A cells either (Fig. 6a). Of interest, expression of dn-PP2A did not markedly affect the basal cell proliferation/viability, but conferred high resistance to rapamycin inhibition of the basal or hsBAFF-stimulated cell proliferation/viability (Fig. 6b, c). Furthermore, we also noticed that the cells expressing dn-PP2A remained sensitive to an Erk1/2 inhibitor, PD98059 (Fig. 6a–c). However, in contrast, overexpression of wild-type PP2A powerfully inhibited the basal or hsBAFF-induced phospho-Erk1/2 and cell proliferation/viability in the presence or absence of rapamycin or PD98059, as detected by Western blotting, as well as cell counting and MTS assay (Fig. 6d–f). Taken together, our data strongly support the idea that rapamycin blocks hsBAFF-induced Erk1/2 activation and cell proliferation/viability via PP2A-dependent mechanism in B cells.

Fig. 6.

Ectopic expression of dominant negative PP2A or wild-type PP2A modulates rapamycin blockage of hsBAFF-induced activation of Erk1/2 and proliferation/viability in B cells. Raji cells, infected with Ad-dn-PP2A, Ad-PP2A and Ad-GFP (as control), respectively, were pretreated with/without rapamycin (100 ng/ml) for 2 h, and then with/without PD98059 (10 μM) for 1 h, followed by treatment with hsBAFF (2.5 μg/ml) for 12 h (for Western blotting) or 48 h (for cell proliferation/viability assay). Total cell lysates were subjected to Western blotting using indicated antibodies (a, d). The blots were probed for β-actin as a loading control. Similar results were observed in at least three independent experiments. The cell proliferation was evaluated by cell counting (b, e) and the cell viability was determined by the MTS assay (c, f). a Rapamycin did not significantly inhibit hsBAFF-induced p-Erk1/2 in Raji/Ad-dn-PP2A cells. b, c Expression of dn-PP2A conferred high resistance to rapamycin inhibition of the basal or hsBAFF-stimulated cell proliferation/viability, and the cells expressing dn-PP2A remained sensitive to PD98059. d–f Overexpression of wild-type PP2A powerfully inhibited the basal or hsBAFF-induced phospho-Erk1/2 (d) and cell proliferation (e)/viability (f) in the presence or absence of rapamycin or PD98059. Results are presented as mean ± SE (n = 6). ^a P < 0.05, difference vs control group; ^b P < 0.05, difference vs 2.5 µg/ml hsBAFF group; ^c P < 0.05, Ad-dn-PP2A group or Ad-PP2A group vs Ad-GFP group

Inhibition of mTOR kinase activity is necessary for rapamycin’s activation of PP2A, leading to suppression of Erk1/2 and cell proliferation/viability in hsBAFF-stimulated B cells

To unveil whether rapamycin’s activation of PP2A, leading to suppression of hsBAFF-induced Erk1/2 activation and B-cell proliferation/viability, is through inhibition of mTOR activity, Raji cells were infected with recombinant adenoviral vectors encoding GFP (Ad-GFP, as control), FLAG-tagged rapamycin-resistant and kinase-active mTOR (S2035T, Ad-mTOR-T), or kinase-dead mTOR-T (S2035T/D2357E, Ad-mTOR-TE) for 24 h, and then pretreated with/without rapamycin (100 ng/ml) for 2 h, followed by stimulation with hsBAFF (2.5 μg/ml) for 12 or 48 h. The functions of the adenoviral constructs were confirmed by determining the expression of FLAG-tagged mTOR mutants and the phosphorylation levels of S6K1 and 4E-BP1. We observed that ectopic expression of FLAG-tagged mTOR-T, but not FLAG-tagged mTOR-TE or GFP, conferred high resistance to rapamycin inhibition of phosphorylation of S6K1 and 4E-BP1 in Raji cells (Fig. 7a), as seen in other cell lines [39, 50]. Of interest, expression of FLAG-mTOR-T, but not mTOR-TE or GFP, rendered high resistance to rapamycin’s inhibitory expression of demethylated-PP2Ac, phospho-PP2Ac and phospho-Erk1/2, leading to reduced cell proliferation/viability in the hsBAFF-stimulated cells (Fig. 7a–c). The results reveal that rapamycin mediates activation of PP2A, thereby inhibiting hsBAFF-induced activation of Erk1/2, as well as cell proliferation/viability, in an mTOR kinase activity-dependent manner.

Fig. 7.

Rapamycin activates PP2A inhibiting hsBAFF-induced Erk1/2 pathway and cell proliferation/viability in B cells in an mTOR kinase activity-dependent manner. Raji cells, infected with Ad-mTOR-T, Ad-mTOR-TE, and Ad-GFP (as control), respectively, were treated with/without 2.5 μg/ml hsBAFF for 12 h (for Western blotting) or 48 h (for cell proliferation/viability assay) following pre-incubation with/without rapamycin (100 ng/ml) for 2 h. Total cell lysates were subjected to Western blotting using indicated antibodies (a). The blots were probed for β-actin as a loading control. Similar results were observed in at least three independent experiments. The cell proliferation was evaluated by cell counting (b) and the cell viability was determined by the MTS assay (c). a Expression of FLAG-tagged mTOR-T, but not FLAG-tagged mTOR-TE or GFP, conferred high resistance to rapamycin’s inhibitory effects on p-S6K1, p-4E-BP1, de-PP2Ac, p-PP2Ac, and p-Erk1/2. b, c Expression of mTOR-T, but not mTOR-TE or GFP, rendered high resistance to rapamycin’s prevention of hsBAFF-induced cell proliferation/viability. Results are presented as mean ± SE (n = 6). ^a P < 0.05, difference vs control group; ^b P < 0.05, difference vs 2.5 µg/ml hsBAFF group; ^c P < 0.05, Ad-mTOR-T group or Ad-mTOR-TE group vs Ad-GFP group

We also confirmed the above findings using RNA interference. As shown in Fig. 8a, lentiviral shRNA to mTOR, but not GFP, silenced the expression of mTOR protein by ~90 % in Raji cells, as detected by Western blotting. Down-regulation of mTOR dramatically decreased the mTOR kinase activity, since the basal or hsBAFF-stimulated phosphorylation of S6K1 at T389, routinely used as an indicator of mTOR kinase activity [21, 22], was not detectable by Western blotting (Fig. 8b). Furthermore, we also noticed that silencing mTOR expression markedly inhibited the phosphorylation of Akt at S473 in the cells in response to hsBAFF and/rapamycin (Fig. 8b), suggesting that both mTORC1 and mTORC2 may be involved in the inhibitory activity of rapamycin in B cells.

Fig. 8.

Down-regulation of mTOR prevents hsBAFF-induced inhibition of PP2A and activation of Erk1/2 as well as proliferation/viability in B cells. Raji cells, infected with lentiviral shRNA to mTOR or GFP (as control), were treated with/without 2.5 μg/ml hsBAFF for 12 h (for Western blotting) or 48 h (for cell proliferation/viability assay) following pre-incubation with/without rapamycin (100 ng/ml) for 2 h. Total cell lysates were subjected to Western blotting using indicated antibodies (a, b). The blots were probed for β-actin as a loading control. Similar results were observed in at least three independent experiments. The cell proliferation was evaluated by cell counting (c) and the cell viability was determined by the MTS assay (d). a Lentiviral shRNA to mTOR, but not to GFP, down-regulated mTOR expression by ~90 % in Raji cells. b Silencing mTOR attenuated expression of hsBAFF-induced p-S6K1, p-Akt, de-PP2A, p-PP2A and p-Erk1/2 in the cells pretreated with/without rapamycin. c, d Down-regulation of mTOR significantly prevented hsBAFF-stimulated B-cell proliferation/viability and strengthened the inhibitory effect of rapamycin. Results are presented as mean ± SE (n = 3–6). ^a P < 0.05 difference vs control group; ^b P < 0.05, difference vs 2.5 µg/ml hsBAFF group; ^c P < 0.05 mTOR shRNA group vs GFP shRNA group

Of importance, down-regulation of mTOR attenuated the expression of hsBAFF-induced demethylated-PP2Ac, phospho-PP2Ac and phospho-Erk1/2 in the cells pretreated with/without rapamycin (Fig. 8c, d), indicating that silencing mTOR increases the PP2A activity, leading to inhibition of Erk1/2 pathway, which mimicked the effects of rapamycin on PP2A-Erk1/2 signaling pathway. Furthermore, as expected, down-regulation of mTOR obviously prevented hsBAFF-stimulated B-cell proliferation/viability and strengthened the inhibitory effect of rapamycin (Fig. 8c, d). Taken together, our data support the concept that PP2A lies downstream of mTOR and is negatively regulated by mTOR. Inhibition of mTOR kinase activity is necessary for rapamycin’s activation of PP2A, leading to suppression of Erk1/2 and cell proliferation/viability in hsBAFF-stimulated B cells.

Discussion

B-cell activating factor of the TNF family is known to be crucial for B cell maturation and survival, and especially increased expression of BAFF in various autoimmune diseases, such as RA, SS and SLE, has recently received more attention [5, 12, 51]. For example, high levels of BAFF contribute to aggressive or neoplastic B cell disorders [16, 17]. Patients suffer from a great quantity of autoantibodies secreted from aggressive B cells, whose root is excess BAFF [8, 12, 51]. However, the signaling pathways that can be activated by BAFF are complex and controversial. It is of great importance to find effective treatments against BAFF-induced autoimmune diseases and other aggressive/neoplastic B-cell disorders. Rapamycin is not only a lipophilic macrolide antibiotic, but also a specific mTOR inhibitor [52]. A series of recent studies have shown that rapamycin can be an effective treatment in both murine lupus models and human SLE [23], and demonstrated that rapamycin could directly alter molecular abnormalities in SLE T cells related to calcium signaling [20, 23]. These findings indicate that rapamycin may act as a pharmacological agent with therapeutic benefits for fighting autoimmune diseases.

In this study, we showed that rapamycin dramatically inhibited cell proliferation and induced cell apoptosis in hsBAFF-stimulated Raji cells, Daudi cells and primary B cells in a concentration-dependent manner. Multiple data have demonstrated that BAFF attenuates apoptosis and stimulates cell proliferation by up-regulating Mcl-1, Bcl-2 and Bcl-xL, down-regulating Bax, enhancing the synthesis of cyclin D2, activating NF-κB and CDK4 in normal and neoplastic B-lymphoid cells [7, 53, 54]. Of note, rapamycin is known to elicit apoptosis by decreasing the expression of Mcl-1, Bcl-2, Bcl-xL, and cFLIP, and by increasing expression of Bax [55–57]. Also, rapamycin inhibits cell proliferation by decreasing the expression of CDKs, cyclins, and Cdc25 and increasing the expression of CDK inhibitors related to the G1/S cell cycle progression in multiple cell types including normal and malignant B cells [43–45]. For instance, rapamycin induces apoptosis of JN-DSRCT-1 cells by increasing the Bax:Bcl-xL ratio with a concomitant activation of caspase-3 [55]; rapamycin induces G1 cell cycle arrest and apoptosis via decreasing the expression of cyclin D2, cyclin D3, and CDK4, and increasing the expression of p27 in Epstein–Barr virus (EBV)-positive B-cell lymphomas [45], and by down-regulating cFLIP, Mcl-1, Bcl-2 and Bcl-xL in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) cells [56] and mantle cell lymphoma (MCL) cells [57]. In this study, although we did not repeatedly test whether rapamycin decreases the expression of anti-apoptotic proteins (e.g., Bcl-2, Bcl-xL, Mcl-1, and cFLIP), we did observe that rapamycin reduced the expression of CDK2, CDK4, CDK6, cyclin A, cyclin D1, and cyclin E (Fig. 4h). Therefore, the findings from us and others suggest that rapamycin inhibits BAFF-stimulated cell proliferation and survival, at least in part through hindering the expression of G1 cell cycle regulatory proteins and anti-apoptotic Bcl-2 family members in normal and neoplastic B-lymphoid cells.

Our recent studies have shown that excessive hsBAFF promotes proliferation and survival in B lymphocytes via activation of mTOR and Erk1/2 signaling network [24, 34]. By inhibiting activation of mTOR pathway, rapamycin can effectively prevent hsBAFF-induced proliferation/viability in primary mouse B lymphocytes [24]. PP2A has been documented as the phosphatase responsible for the dephosphorylation of S6K1 and 4E-BP1, and activation of PP2A by rapamycin is linked to dephosphorylation of S6K1 and 4E-BP1 [35]. PP2A also negatively regulates Erk1/2 pathway through dephosphorylation of the Erk1/2 protein [25]. We have uncovered that hsBAFF activates Erk1/2, in part via inhibition of PP2A, in normal and neoplastic B-lymphoid cells [34]. Putting all data together, we postulated that a cross-talk may occur between mTOR and PP2A-Erk1/2 pathways in B cells in response to BAFF, i.e., BAFF stimulation of mTOR may repress PP2A, resulting in activation of Erk1/2, which may be prevented by rapamycin. This study, for the first time, presents evidence that rapamycin inhibited excess hsBAFF-stimulated cell proliferation and survival indeed by activation of PP2A, resulting in inhibition of Erk1/2 pathway. Furthermore, the effect of rapamycin on PP2A-Erk1/2 signaling was by suppression of mTOR kinase activity in normal and neoplastic B-lymphoid cells. Our data underscore that rapamycin has an ability to prevent excessive BAFF from activation of mTOR-mediated PP2A-Erk1/2 signaling pathway in the B cells.

PP2A has been widely recognized as a key regulator for cell proliferation, migration, and survival [25–27, 41]. PP2A consists of a catalytic subunit (PP2Ac), an A regulatory subunit (PP2A-A), and a number of B regulatory subunits (PP2A-B) [42]. Accumulating data have shown that the phosphatase activity of PP2Ac is modulated by its association with PP2A-A, -B regulatory subunits [42]. Also the activity can be turned “on” and “off” by post-translational modification such as phosphorylation and carboxyl methylation of PP2Ac [58, 59]. Multiple studies have described that disturbances in the expression and/or function of PP2A are linked to autoimmune diseases [27, 30, 31]. For example, cellular levels of the catalytic subunit of PP2A are abnormally high in patients with SLE [28], whereas PP2A-Bβ levels are abnormally low in some patients with SLE [60]. Fernandez et al., by applying rapamycin to SLE patients refractory to standard treatments, have found that patients respond well to rapamycin, leading to a decrease in disease activity and prednisone requirement, pointing to the applicability of rapamycin in the context of SLE [20]. In addition, as PP2A has been linked to rapamycin’s sensitivity [37], this let us to investigate the effect of rapamycin on PP2A activity in B-cell proliferation/viability triggered by hsBAFF. In this study, we did not observe that rapamycin altered cellular protein expression of the catalytic subunit (PP2Ac) and the regulatory proteins (PP2A-A and PP2A-B) (Fig. 2a). However, we found that hsBAFF-induced robust expression of demethylated-PP2Ac and phospho-PP2Ac (Tyr307) was attenuated by rapamycin concentration-dependently in Raji cells, Daudi cells and primary B lymphocytes (Fig. 2a). This is further evidenced by the in vitro Ser/Thr phosphatase assay, showing that rapamycin prevented hsBAFF from inhibition of PP2A activity (Fig. 2b). These data indicate that rapamycin activates the phosphatase activity of PP2A at least by inhibiting hsBAFF-elevated demethylation and phosphorylation of PP2Ac, two events responsible for PP2A inactivation [42]. Concurrently, rapamycin also significantly blocked hsBAFF-induced remarkable phosphorylation of Erk1/2. Collectively, these observations suggest that rapamycin may activate PP2A and inactivate Erk1/2 signaling, which is associated with rapamycin’s inhibition of hsBAFF-stimulated proliferation and survival in normal and B-lymphoid cells.

Erk1/2 lies downstream of the Ras-Raf-MKK cascade [61]. It is well known that PP2A negatively regulates Erk1/2 pathway through dephosphorylating and inactivating both MKK1/2 and Erk1/2 proteins [25]. To test the hypothesis that rapamycin prevents hsBAFF-induced Erk1/2 activation and proliferation/viability via PP2A-dependent mechanism in B cells, pharmacological/genetic inhibition or rescue studies for Erk1/2, MKK1 or PP2A were carried out, respectively. We found that pretreatment with PD98059 (a selective inhibitor of MKK1/2, upstream of Erk1/2), down-regulation of Erk1/2 using lentiviral shRNA to Erk1/2, expression of dominant negative MKK1, or overexpression of wild-type PP2Ac potentiated rapamycin’s suppression of hsBAFF-induced phosphorylation of Erk1/2 and B-cell proliferation/viability, whereas expression of constitutively active MKK1, inhibition of PP2A by okadaic acid, or expression of dominant negative PP2A resulted in the robust phosphorylation of Erk1/2 and conferred potent resistance to rapamycin inhibition of hsBAFF-stimulated cell proliferation/viability in the cells. Furthermore, we also noticed that the cells expressing dn-PP2A were able to remain sensitive to MKK1/2 inhibitor PD98059. In contrast, overexpression of wild-type PP2A dramatically inhibited the basal or hsBAFF-induced phosphorylation of Erk1/2 and cell proliferation/viability in the presence or absence of rapamycin or PD98059. The results support a model in which rapamycin inhibits critical survival signals including MKK1/2 and Erk1/2, through activation of PP2A, leading to reduction of BAFF-stimulated cell proliferation and survival in normal and neoplastic B-lymphoid cells.

Cell proliferation is associated with cell cycle progression [43]. Recent growing evidence have highlighted that the activated Erk1/2 stimulates the transcription of CDK4, CDK6 and cyclin D genes [46]. Pharmacological inhibition of Erk1/2 potently decreases CDK2, CDK4, cyclin D1 and cyclin E expression, and increases the levels of CDK inhibitors p21 and p27 [47, 48]. Therefore, we reasoned that rapamycin’s blockage of hsBAFF-activated Erk1/2 pathway contributes to decreased B-cell proliferation/viability by altering the expression of cell cycle regulatory proteins. To answer the question, Raji cells, infected with lentiviral shRNA to Erk1/2 or GFP, were pretreated with/without rapamycin (100 ng/ml) for 2 h and then stimulated with/without hsBAFF (2.5 μg/ml) for 12 h. The results showed that expression of cyclin A, cyclin E, cyclin D1, CDK2, CDK4 and CDK6 was obviously blocked, and concurrently two CDK inhibitors p21 and p27 were strongly upregulated in Erk1/2-silenced Raji cells stimulated with or without hsBAFF. Importantly, silencing Erk1/2 enhanced the effects of rapamycin on hsBAFF-induced expression of cyclin A, cyclin E, cyclin D1, CDK2, CDK4 and CDK6, in spite of failure to further increasing the expression of p21 and p27 in the presence of rapamycin. Thus, our data indicate that rapamycin’s inhibition of Erk1/2 pathway contributes to down-regulating cyclin A, cyclin E, cyclin D1, CDK2, CDK4 and CDK6, and up-regulating p21 and p27, leading to inhibition of hsBAFF-stimulated B-cell proliferation.

Rapamycin is a well-known mTOR inhibitor [21, 22]. However, studies have also shown that rapamycin represses differentiation of C2C12 cells [50], which is mTOR kinase activity-independent, although this remains controversial [50, 62]. This prompted us to study whether rapamycin activates PP2A leading to inactivation of Erk1/2 and reduction of cell proliferation/viability in the B cells in response to hsBAFF in an mTOR kinase activity-dependent manner. To this end, the recombinant adenoviruses expressing FLAG-tagged rapamycin-resistant and kinase-active mTOR (Ad-mTOR-T) or kinase-dead mTOR-T (Ad-mTOR-TE) was utilized. We found that rapamycin failed to inhibit hsBAFF-induced inactivation of PP2A, activation of Erk1/2 and cell proliferation/viability in Ad-mTOR-T-transfected cells, but not in control cells (Ad-GFP- or Ad-mTOR-TE-infected cells), suggesting an mTOR-dependent mechanism involved. This is further supported by the findings that silencing mTOR resulted in activation of PP2A, leading to inhibition of Erk1/2 and cell proliferation/viability, and this was potentiated by rapamycin. Collectively, these findings reveal that inhibition of mTOR is required for rapamycin’s activation of PP2A and inactivation of Erk1/2 in B cells. Furthermore, in this study, we also noticed that silencing mTOR obviously blocked the phosphorylation of Akt at S473 in Raji cells in response to hsBAFF, suggesting that both mTORC1 and mTORC2 may be involved in the inhibitory activity of rapamycin. Our results provide an expanded conceptual view of rapamycin’s effects on BAFF-dependent survival signals, which should contribute to a better understanding of rapamycin’s effective treatment for excessive BAFF-induced aggressive or neoplastic B-cell disorders associated with autoimmune diseases.

In summary, here we have shown that rapamycin inhibited excess hsBAFF-stimulated cell proliferation and survival by activation of PP2A and inactivation of Erk1/2 pathway in normal and neoplastic B-lymphoid cells. Further, the effect of rapamycin on PP2A-Erk1/2 signaling pathway was through inhibition of mTOR activity in the cells. Our data highlight that rapamycin may be exploited for prevention of excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.

Abbreviations

4E-BP1

Eukaryotic initiation factor 4E binding protein 1

Akt

Protein kinase B (PKB)

BAFF

B-cell activating factor of the TNF family

BLyS

B lymphocyte stimulator

BCMA

B-cell maturation antigen

CDK

Cyclin-dependent kinase

Erk1/2

Extracellular signal-related kinases 1/2

MAPK

Mitogen-activated protein kinase

MKK

Mitogen-activated protein kinase kinase

mTOR

Mammalian target of rapamycin

PP2A

Protein phosphatases 2A

S6K1

Ribosomal protein S6 kinase 1

SLE

Systemic lupus erythematosus

TACI

Transmembrane activator and cyclophilin ligand interactor

TALL-1

TNF and apoptosis ligand-related leukocyte-expressed ligand1

THANK

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

Conflict of interest

The authors declare that they have no conflict of interest.