Background
Endoplasmic reticulum (ER) calcium homeostasis plays an important role in the control of cell activation [
1,
2]. Calcium release from the ER by the second messenger D-
myo-inositol-1,4,5-
tris- phosphate (IP3) upon activation of receptors such as the B-cell or chemokine receptors, combined with ensuing calcium influx across the plasma membrane (store operated calcium entry, SOCE), leads to the activation of key calcium-dependent enzymes involved in cell activation such as various protein kinase C isoforms, calcineurin or calmodulin dependent kinases [
2‐
6]. Because these enzymes critically modulate the activity of transcription factors such as NF-κB, NF-AT or AP1, calcium signalling, initiated by calcium release from the ER, plays important roles in B cell survival, maturation and activation. In addition, normal ER calcium homeostasis is also required for the post-translational modifications/chaperoning of nascent immunoglobulin molecules taking place in the organelle, a phenomenon that is also involved in the regulation of B cell maturation and plasma cell survival [
7‐
9].
Because calcium accumulation into the endoplasmic reticulum (ER) is accomplished by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA-type calcium pumps) that generate a strong calcium concentration gradient between the cytosol (low nanomolar) and the ER lumen (high micromolar), precisely regulated SERCA activity is essential for normal cell function and survival [
10]. SERCA-type calcium pumps are encoded by three genes (SERCA1, 2 and 3) that give rise by alternative splicing to several isoenzymes. Whereas SERCA1 is expressed in skeletal muscle, and the SERCA2a isoform is found in cardiac muscle, SERCA2b expression is ubiquitous. In several cell types such as T lymphocytes, myeloid cells, megakaryocytes and platelets, as well as in colon and gastric epithelial cells SERCA2b is co-expressed with SERCA3 [
11‐
18]. Whereas the basic biochemical function (
i.e. calcium transport into the ER) is shared among SERCA2 and SERCA3 isoenzymes, the calcium affinity of SERCA3 is significantly inferior to that of SERCA2b [
10,
19‐
22]. The co-expression of SERCA2b and SERCA3 within the same cell is thus involved in the fine regulation of the calcium uptake characteristics of the ER in a cell type-dependent manner.
Epstein-Barr virus, a human gammaherpesvirus can immortalize primary naïve B lymphocytes, leading to the establishment of permanently growing lymphoblastoid cell lines (LCL). In addition, the virus is etiologically involved in the formation of several types of malignancies such as Burkitt's lymphoma, lymphomas of immunocompromised individuals, some NK/T lymphomas, lymphomatoid granulomatosis, pyothorax-associated lymphoma, Hodgkin's lymphoma, as well as gastric and nasopharyngeal carcinomas (for a comprehensive review of EBV biology see [
23]). EBV-induced immortalization of B cells into LCLs, as well as malignant transformation are linked to the viral latent membrane protein-1 (LMP-1), the expression of which is under the control of the EBNA2 virus encoded protein. LMP-1 displays great functional homology with activated CD40 normally expressed on B cells [
23,
24]. LMP-1 constitutively activates a variety of signalling cascades including NF-κB, ERK, JNK, and p38 MAP kinase, as well as the Akt-PI3K pathway, is responsible for the phenotypic changes observed during conversion of B cells into LCLs, and is a
bona fide transforming oncogene in various experimental systems [
23,
25].
EBV infection, as well as normal lymphocyte activation have been shown to lead to increased resting cytosolic calcium concentration [
26,
27]. Calcium release from the ER into the cytosol through IP3 receptor calcium channels is essential for B cell function at various stages of B cell differentiation [
27‐
29]. Calcium-dependent cell activation is initiated by calcium release from the ER through IP3 receptors [
1,
2], and is greatly amplified by subsequent calcium influx from the extracellular space through Orai1-type store-operated calcium channels that are opened by decreased calcium levels in the ER lumen [
4,
30]. Because calcium release is critically dependent on ER calcium accumulation by SERCA-type enzymes, and because SERCA activity is instrumental in the shaping of the amplitude, the intensity and the duration of cellular calcium signals [
31‐
33] and therefore of cell activation [
34‐
36], in this work we investigated endoplasmic reticulum calcium pump expression and function in various EBV-infected B cells, in cells that express transgenic key EBV proteins such as LMP-1, and compared their effects to normal B cell activation
in situ.
Discussion
The EBV life cycle in B cells is critically regulated by cellular calcium signalling: protein kinase C and calmodulin dependent kinase activation are key factors that determine the transition from latency to virus replication and host cell lysis. In experimental settings this is reflected by the strong capacity of agents that increase cytosolic calcium levels to induce the lytic cycle in latently infected B cells. Indeed, calcium ionophores, such as A23187 or ionomycin, are strong inducers of EBV reactivation [
42].
Endoplasmic reticulum calcium homeostasis is also involved in several mechanisms essential for normal B lymphocyte function at various stages of differentiation. Appropriate calcium signalling is necessary for pre-B cell receptor, as well as B-cell receptor-dependent intracellular signalling and lymphocyte selection/survival, differentiation and antigen-dependent activation [
27,
28,
43]. In addition, plasmocytes, that are fully differentiated, antibody secreting B cells, build up large amounts of endoplasmic reticulum, as this organelle is involved in immunoglobulin synthesis and maturation [
44‐
46].
Although the importance of ER calcium homeostasis and signalling in the control of EBV replication is clearly established in the literature [
42,
47], data on the effect of EBV on ER calcium homeostasis, and in particular on SERCA expression and function were lacking. In this work we show, for the first time, that SERCA expression and activity are modified by EBV in B cells in type III latency by LMP-1. A marked decrease of SERCA3 expression could be observed in various Burkitt's lymphoma cell lines infected with the B95–8 EBV strain that displays full immortalizing capacity and expresses LMP-1 and EBNA2, whereas the non-immortalizing P3HR-1 virus strain that lacks LMP-1 expression due to deletions in the sequence of the EBNA2 viral transactivator [
38] was without effect. Importantly, down-modulation of SERCA3 expression could also be induced by the expression of LMP-1
in trans, in the absence of any other viral elements, such as EBNA2 or EBNA3A, 3B or 3C, in various inducible expression systems, indicating that LMP-1 alone is sufficient for SERCA3 down-modulation. LMP-1 expression led to increased resting cytosolic calcium levels in accordance with previous observations [
48] and, importantly, to increased calcium storage in the ER as detected by thapsigargin-induced calcium release, indicating that LMP-1 can induce functional changes in the ER calcium homeostasis of the host cell. Unlike during induction of the expression of LMP-1 cDNA, resting cytosolic calcium levels were not significantly increased in chronically EBV-infected cells, probably due to secondary compensatory mechanisms, such as increased SERCA2 expression, that occur during the establishment of latency, or due LMP-2A that has been shown to blunt calcium mobilisation in B cells [
49]. However, SERCA3 down-modulation and enhanced ER calcium storage could be observed, both in Burkitt's lymphoma cell lines infected with the prototypical, immortalizing B95–8 EBV strain and in EBV-free cells expressing only an LMP-1 transgene. These data taken together show, that EBV modifies SERCA expression as well as functional calcium accumulation into the ER of latently infected B cells, by the LMP-1 viral oncoprotein. LMP-1 activates several signalling pathways including NF-κB, STATs, ERK, JNK, p38 MAPK, as well as PI3K/Akt. Due to the simultaneous activation and cross-talk among these regulatory mechanisms, the identification of the direct molecular mechanism of LMP-1-induced SERCA3 down-regulation requires further work. However, our data show, for the first time, that the modulation of ER calcium homeostasis is part of the cell activation program induced by LMP-1.
The functional consequences of the modulation of SERCA expression by EBV in B cells are complex. The various protein types involved in cellular calcium homeostasis function in a highly interconnected manner, due to the fact that the transport activity of various SERCA and PMCA enzymes, as well as the opening probability of IP3 receptor calcium channels are regulated by the calcium concentration of the cytosol or of the ER lumen. This leads to positive, as well as negative feedback regulation that often generate oscillatory cellular calcium signals, the frequency and amplitude of which can selectively affect the activation of target proteins such as PKC or calcineurin [
50‐
54]. Computer modelling of the highly dynamic interplay of calcium pumps, channels and target enzymes, coupled to experimental observation made on cells in which SERCA3 expression was ablated, led to the conclusion that SERCA3 function is particularly important for the shaping of the characteristics (amplitude, frequency and duration) of calcium oscillations [
31‐
33,
55,
56]. For instance, it has been shown, that the loss of SERCA3 expression leads to altered glucose-induced calcium oscillations and electrical activity that control insulin secretion in the β cells of the endocrine pancreas [
55,
57]. The calcium affinity of SERCA3 (KCa
2+ ~ 1.2 μM) is significantly lower than that of the simultaneously expressed SERCA2b isoenzyme (KCa
2+ ~0.2 μM) [
19,
21,
22,
58]. Because the variations of cytosolic calcium levels during cell activation in the vicinity of the ER lie within the range in which SERCA3 activity is modulated by calcium, the amount of SERCA3 expressed in a cell has a major impact on the shape and frequency of calcium signals, and therefore can determine the state of activation of the cell, whereas SERCA2b function is more probably involved in constitutive calcium uptake in the ER, linked to "housekeeping" functions, as this enzyme is probably almost fully active already at resting cytosolic calcium levels.
The results presented in this paper on increased intracellular calcium pool size in the presence of EBV or LMP-1 expression are compatible with the observed decrease of SERCA3 expression. It may be hypothesised that decreased SERCA3 expression reflects the loss or depletion of a lower affinity or leakier intracellular ER sub-compartment, leading to calcium accumulation into a higher affinity, SERCA2b associated ER pool containing more calcium releasable by SERCA inhibition. The exact structural and functional relationship between SERCA2 and SERCA3-associated intracellular calcium pools in B cells remains, however, to be determined.
Enhanced calcium accumulation induced by LMP-1 may be involved in the establishment of viral latency. Because the EBV lytic cycle can be induced by increased cytosolic calcium levels (as observed with calcium ionophores or during B-cell receptor activation [
42,
47,
59]), a more stringent calcium sequestration in the ER by the higher affinity SERCA2b isoform may blunt calcium signals that would otherwise lead to the induction of the lytic cycle. Moreover, SERCA3 has been shown earlier to be associated with the IP3-sensitive sub-compartment of the ER in platelets [
60]. It is therefore tempting to hypothesize that the selective down-regulation of SERCA3 expression in EBV infected B cells may correspond to decreased calcium uptake in the IP3-sensitive intracellular calcium pool with calcium uptake being redirected towards a SERCA2-dependent sub-compartment of the ER. This may lead to a state of chronic, constitutive activation of the cells and their desensitisation for further IP3-mobilizing signals. Although the description of the detailed mechanisms of the involvement of ER calcium homeostasis in EBV replication and latency require further studies, our work shows, for the first time, that ER calcium homeostasis is modulated by EBV
via LMP-1, and opens new avenues in the understanding of the interactions taking place between EBV and the infected lymphocyte.
SERCA3 down-modulation was observed also during normal B lymphocyte activation in lymph node, as well as in mucosa-associated lymphoid follicles. In these tissues the mantle zone represents small resting B lymphocytes admixed with sparse helper and suppressor T cells, whereas in germinal centers where antigen stimulation of B cells by follicular dendritic cells takes place, centrocytes and centroblasts accumulate [
43]. As shown in this work by immunohistochemistry, whereas small resting B lymphocytes in the mantle zone express SERCA3 abundantly, SERCA3 expression markedly decreases during antigenic stimulation in germinal centers, indicating that SERCA3 down-modulation is part of the normal B lymphocyte activation program, where this phenomenon is related to antigen-driven activation and blastic transformation of B cells. Although EBV-immortalized lymphoblastoid cells and normal germinal center B lymphoblast display phenotypic differences, both cell types are activated, proliferating B cells. Down-modulation of SERCA3 expression by EBV should be considered therefore as a previously unknown virus-induced phenomenon taking place during establishment of type III latency and immortalization that mimics a normal step of B cell activation.
Acknowledgements
We express special thanks to Prof. Neville Crawford (Department of Biochemistry, the Hunterian Institute, London, U.K.) for giving us the PLIM430 hybridoma, to Dr Bettina Kempkes (Institute of Clinical Molecular Biology, GSF National Research Centre for Environment and Health, Munich, Germany) for the BL41/K3 cells, and to Mr. Patrice Castagnet for excellent technical help. This work was supported by Inserm, the Association pour la Recherche sur le Cancer, the Ligue contre le Cancer, Fondation de France, the Association Laurette Fugain and by the Hungarian Academy of Sciences Grant OTKA T046814 (to T.K.)
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
OD and BP designed and performed research, collected, analysed and interpreted data and wrote the manuscript. CC interpreted data, AA performed research, collected and analyzed data, IJ, JPB, MR and TK contributed vital reagents and interpreted data. All authors read and approved the final manuscript.