STIM- and Orai-mediated calcium entry controls NF-κB activity and function in lymphocytes

Abstract

The central role of Ca²⁺ signaling in the development of functional immunity and tolerance is well established. These signals are initiated by antigen binding to cognate receptors on lymphocytes that trigger store operated Ca²⁺ entry (SOCE). The underlying mechanism of SOCE in lymphocytes involves TCR and BCR mediated activation of Stromal Interaction Molecule 1 and 2 (STIM1/2) molecules embedded in the ER membrane leading to their activation of Orai channels in the plasma membrane. STIM/Orai dependent Ca²⁺ signals guide key antigen induced lymphocyte development and function principally through direct regulation of Ca²⁺ dependent transcription factors. The role of Ca²⁺ signaling in NFAT activation and signaling is well known and has been studied extensively, but a wide appreciation and mechanistic understanding of how Ca²⁺ signals also shape the activation and specificity of NF-κB dependent gene expression has lagged. Here we discuss and interpret what is known about Ca²⁺ dependent mechanisms of NF-kB activation, including what is known and the gaps in our understanding of how these signals control lymphocyte development and function.

Figure 1. (Graphical Abstract): Antigen receptor induced Ca²⁺ dynamics tune the patterns. of transcriptional activation.

This schematic proposes how variations in antigen receptor induced Ca²⁺ dynamics elicit distinct patterns of gene expression depicted by a heatmap of differential gene expression. Work over the past 30 years has established that the strength of antigen receptor stimulation is encoded as quantitatively distinct patterns of Ca²⁺ signaling, and that these can each initiate a distinct transcriptionally driven fate of lymphocytes by activating Ca²⁺ dependent transcription factors. The key pro-inflammatory transcription factors NF-κB and NFAT, which exhibit differences in Ca²⁺ sensitive activation, decode differences in the strength of antigen stimulation into distinct patterns of transcriptional activation. High affinity antigen binding causes sustained STIM/Orai-dependent Ca²⁺ entry and these signals activate both NF-κB and NFAT. Intermediate antigen affinity/avidity interactions cause distinct patterns of signaling, possibly Ca²⁺ oscillations, that preferentially activate NF-κB and depending on the frequency can potentially activate NFAT. Low affinity/avidity interactions that cause a significant but transient elevation in cytoplasmic Ca²⁺ leading to the selective activation of NF-κB signaling and NF-κB dependent target gene expression. Finally, in the absence of Ca²⁺ entry, low amplitude, transient elevations in cytoplasmic Ca²⁺ may be insufficient for the activation of either NF-κB or NFAT.

Figure 2:. Ca²⁺ regulated checkpoints in canonical NF-κB signaling in lymphocytes

A schematic representation of known mechanisms by which Ca²⁺ regulates the activation of NF-κB dependent gene expression. In resting lymphocytes, homo- and heterodimers composed of NF-κB p50, p65, and c-Rel proteins are sequestered in the cytoplasm by IκB proteins including IκBα. BCR or TCR engagement activates a series of protein tyrosine kinases (PTKs) that subsequently activate PLCγ isoforms, cleaving PtdIns(4,5)P2 to generate InsP3 (IP3) and DAG. IP3 binds to IP3 receptors (IP3R) channels allowing release of Ca²⁺ from the endoplasmic reticulum (ER). The resulting decrease in ER [Ca²⁺] causes oligomerization of STIM1 proteins and activation of plasma membrane Orai1 channels facilitating extracellular Ca²⁺ entry and a significant or sustained elevation in cytoplasmic Ca²⁺. In B cells, Ca²⁺ and DAG activation of PKCβ and in T cells, DAG activation of PKCθ controls assembly and activity of the CARMA-BCL10-MALT1 (CBM) complex, which is required for antigen receptor induced activation of the IKK complex (NEMO-IKKα-IKKβ). Furthermore, PKCα, the CaM dependent phosphatase Calcineurin (CaN), and calmodulin (CaM)-dependent kinase II (CaMKII) each also regulate CBM complex formation by controlling the phosphorylation levels of these proteins. Ca²⁺ also plays a negative regulatory role via CaM activation that can directly bind to BCL10 to disrupt the CBM complex, activate CaMKII that phosphorylates BCL10 on S138 or binds to NF-κB to block nuclear translocation (all depicted by red lines). NEMO recruitment to the CBM complex facilitates full activation of the IKK complex, which phosphorylates IκB proteins, marking them for ubiquitylation and proteasomal degradation. Both p65 and c-Rel can undergo Ca²⁺ dependent (e.g. Ser536 phosphorylation, CaM binding) and independent post-translational modifications that regulate their nuclear localization, transactivation, and target gene binding potential. Thus, Ca²⁺ dependent mechanisms that regulate the IκB protein degradation and expression and those that modify the post-translational landscape of NF-κB proteins cooperate to regulate gene expression.

Figure 3:. STIM/Orai dependent regulation of nTreg differentiation by NF-κB and NFAT signaling.

A schematic and simplified representation depicting Ca²⁺ dependent mechanisms that control the generation of natural regulatory T cells from CD4SP thymocytes. The upstream events leading to full NF-κB activation are described in Fig.2. NFAT is activated by Ca²⁺-dependent calcineurin (Cn) mediated dephosphorylation. Intermediate to high affinity interactions with self-antigen drive the upregulation of c-Rel (1) and survival (2) of a select proportion of T cells through NFAT and NF-κB dependent mechanisms. A population of c-Rel expressing thymocytes (nTreg progenitors) then upregulate CD25 expression (3) to generate nTreg precursors through Ca²⁺ and c-Rel dependent mechanisms. Sufficient binding of cytokines including IL2 drive upregulation of Foxp3 (4) in nTreg precursors generating functional CD4+c-Rel+CD25+Foxp3+ T cells capable of inhibiting autoreactive or excessive inflammatory immune reactions.

Figure 4:. STIM/Orai and NF-κB dependent regulation of B lymphocyte activation, survival, and function.

BCR triggered STIM/Orai dependent Ca²⁺ entry critically regulates B cell activation and survival. In the absence of BCR induced Ca²⁺ signals or strong co-stimulation, B cells undergo apoptosis through undefined but likely NF-κB dependent mechanisms. Appropriate BCR and costimulatory activation rescues cells from apoptosis and initiates their growth and metabolic programing. STIM/Orai dependent transcriptional induction of key metabolic transcriptional regulators and NF-κB/NFAT target genes Myc, Hif1a, and Irf4 is critical to the survival and function of mature B cells. Upregulation of Myc, Hif1-α, and Irf4 orchestrates the induction of critical glycolytic intermediates and facilitate the reprogramming of lymphocyte metabolism from oxidative phosphorylation to glycolysis. These metabolic changes support rapid cell growth, DNA replication, and cell division. Additional roles for STIM/Orai dependent regulation of B cell function are evident in regulatory B cells (Bregs). Bregs are defined by their expression of suppressive cytokines including IL-10 and TGF-β and in the absence of STIM/Orai mediated calcium entry exhibit defective NF-κB and NFAT dependent Il10 expression.