Calcium signalling and cell-fate choice in B cells

Abstract

Alterations in the cytosolic concentration of calcium ions (Ca2+) transmit information that is crucial for the development and function of B cells. Cytosolic Ca2+ concentration is determined by a balance of active transport and gradient-driven Ca2+ fluxes, both of which are subject to the influence of multiple receptors and environmental sensing pathways. Recent advances in genomics have allowed for the compilation of an increasingly comprehensive list of Ca2+ transporters and channels expressed by B cells. The increasing understanding of the function and regulation of these proteins has begun to shift the frontier of Ca2+ physiology in B cells from molecular analysis to determining how diverse inputs to cytosolic Ca2+ concentration are integrated in specific immunological contexts.

Figure 1. Ca²⁺ physiology in B cells at rest and during InsP₃-mediated Ca²⁺ signal

A: In "resting" B cells, cytoplasmic Ca²⁺ homeostasis is maintained primarily through the actions of plasma membrane Ca²⁺ ATPase (PMCA) and sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) active transporters or pumps. Na⁺/Ca²⁺ exchange is limited due to lack of cytoplasmic Ca²⁺, and plasma membrane channels are primarily in the closed state. B: A receptor-mediated (inositol-1,4,5-trisphosphate (IP₃))-dependent calcium signal is initiated through binding of IP₃ to IP₃R proteins in the endoplasmic reticulum (ER) membrane. Opening of IP₃R channels allows Ca²⁺ to enter the cytoplasm from ER Ca²⁺ stores. Once ER Ca²⁺ stores are sufficiently depleted, ER Stromal interaction molecule (STIM) family proteins are activated to move into proximity to, and open, Ca²⁺-release activated channels (CRAC channels) in plasma membrane microdomains. Notable aspects of cell physiology in the context of an active Ca²⁺ signal include: (1) plasma membrane potential provides a significant driving force for Ca²⁺ entry through ion channels; (2) active Ca²⁺ transport activities (sarcoplasmic reticulum/endoplasmic reticulum Ca²⁺ ATPase (SERCA), plasma membrane Ca²⁺ ATPase (PMCA), and Na⁺/Ca²⁺ exchange) increase relative to the resting state in response to the increased availability of Ca²⁺ on their pumping cytosolic surfaces; and (3) Na⁺ entry into the cytoplasm may significantly influence cytosolic Ca²⁺ concentration depending on local changes in Na⁺ concentrations and the relative flow of Ca²⁺ through Ca²⁺ selective ion channels, non-selective cation channels (G), and reverse mode Na⁺ /Ca²⁺ exchange (that is, Ca²⁺ transported to the cytosol from the extracellular environment in exchange for intracellular Na⁺)–.

Figure 2. Biochemical events in a BCR signaling complex leading to amplified PLCγ2 activation

A: Recruitment and phosphorylation (yellow arrows and dots) of B-cell linker (BLNK) protein within the signalosome of the B-cell receptor (BCR) generates binding sites and recruitment (green arrows) of key BCR effectors VAV, GRB2 (growth-factor-receptor-bound protein 2), phospholipase Cg2 (PLCγ2) and Bruton's tyrosine kinase (BTK). B: P110 Phosphoinositide-3-kinase (PI3K) activation occurs through several parallel pathways (black arrows), and leads to local phosphorylation (yellow arrow) of phosphatidylinositol-4,5-bisphosphate to phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P₃). This effect is amplified in part through recruitment (green arrow) of the p85 subunit of PI3K to the CD19 complex. C: Recruitment of BTK to PtdIns(3,4,5)P₃ induces its transphosphorylation by lyn (small yellow arrows), and initiates a positive feedback loop (large yellow arrows): association of BTK with phosphatidyl inositol-4-phosphate 5-kinase (PI5K) leads to increased production of phosphatidylinositol-4,-5- bisphosphate (PtdIns(4,5)P₂) and so increased substrate for PtdIns(3,4,5)P₃production; enhanced PtdIns(3,4,5)P₃ leads to increased BTK recruitment (with additional PI5K) and BTK activation. The overall signal is read out through BTK-dependent tyrosine phosphorylation and activation of PLCγ2 (red arrows), which hydrolyzes (black arrows) locally available and newly synthesized PtdIns(4,5)P₂ to IP₃ and diacylglycerol (DAG).

Figure 3. Surface-receptor mechanisms for modulation of BCR-induced Ca²⁺ signals

The CD19 complex is a positive regulatory receptor. Its active recruitment of into B-cell receptor (BCR) signaling complexes acts to enhance p110 phosphoinositide-3-kinase (PI3K) activation during BCR signaling. FcγRIIb1 and CD22 are both negative regulators of BCR-mediated calcium signals. When actively recruited into BCR signaling complexes, these receptors both act by recruiting the SH2-containing tyrosine phosphatase-1 (SHP1) and the SH2-containing inositol-5'-phosphatase (SHIP). However, the majority of the impact of FcγRIIb1 on Ca²⁺ signaling is mediated through the SHIP-dependent attentuation of PI3K signaling, while CD22 appears to have its most significant effect on Ca²⁺ signaling through SHP-1-dependent enhancement of PMCA-mediated Ca²⁺ export.

Figure 4. Mechanisms for regulation of cytosolic Ca²⁺ in B cells

Pathways with known or potential ability to influence cytosolic Ca²⁺ in B-cells are grouped by colour according to their mechanism of influence.

Figure 5. Membrane potential modulation of Ca²⁺ signals

Membrane potential can be differentially and dynamically regulated through K⁺ channel activity and TRPM4/TRPM5 monovalent cation channel activity Top panel: In resting cells, membrane potential is set at a highly negative potential through the activity of voltage–operated K⁺ channels. The negative membrane potential is generated through the outward movement of positively charged K⁺ ions, leaving unpaired intracellular negative charges (not shown). TRPM4/TRPM5 channels are thought to be closed in resting cells. Middle panel: When a cell stimulus leads to activation of the store-operated Ca²⁺ entry (SOCE) pathway, opening of highly Ca²⁺-selective CRAC channels allows Ca²⁺ to pass across the plasma membrae. The current carried by CRAC channels is very small, and thus the entering positively charged Ca²⁺ ions have little direct influence on the membrane potential. However, the rise in cytosolic Ca²⁺ resulting from CRAC channel opening may influence the gating of both Ca²⁺-activated K⁺ channels and TRPM4/TRPM5 channels. Bottom left panel: In cells expressing significant numbers of Ca²⁺-activated K⁺ channels, their activation serves to maintain a highly negative membrane potential. Thus, the cell maintains a strong sustained driving force for Ca²⁺ entry and a large magnitude sustained Ca²⁺ signal results. Bottom right panel: In cells expressing significant numbers of TRPM4/TRPM5 monovalent cation channels, their activation allows Na+ entry, thus providing cytosolic positive ions to neutralize the unpaired internal charges. This diminishes the negative membrane potential, thereby attenuating the driving force for Ca²⁺ entry, and resulting in a reduced magnitude Ca²⁺ signal. Beyond Ca²⁺-dependent activation of K⁺ and TRPM4/TRPM5 channels, the activity of these channels is variously accessible to receptor mediated stimuli, metabolic mediators, and environmental influences that modulate their gating properties, providing multiple mechanisms through which dynamic regulation of membrane potential could influence B-cell Ca²⁺ signaling.

Figure 6. Potential mechanisms for Ca²⁺-dependent modulation of B-cell fate determination

Based on present models for Ca²⁺-dependent regulation of transcription factors such as Nuclear Factor of Activated T-cells (NFAT) and Nuclear Factor kb (NF-κB), membrane potential modulation and direct Ca²⁺ entry through non-SOCE pathways are predicted to have the capacity to significantly alter B-cell-fate choice. A: Strong B-cell receptor (BCR) activation fully activates both NF-κB (through inhibitor of kb (IκB) degradation) and NFAT (through calcineurin phosphatase activation) pathways. B: Modulation of membrane potential attenuates late Ca²⁺ entry, selectively influencing NFAT activation. C: A weak but sustained BCR activation signal activates only the NFAT pathway. D: Late activation of a direct Ca²⁺ entry pathway restores NF-κB pathway activation despite a weak BCR activation signal.