Different agonists recruit different stromal interaction molecule proteins to support cytoplasmic Ca2+ oscillations and gene expression.

Stimulation of cells with physiological concentrations of calcium-mobilizing agonists often results in the generation of repetitive cytoplasmic Ca(2+) oscillations. Although oscillations arise from regenerative Ca(2+) release, they are sustained by store-operated Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels. Here, we show that following stimulation of cysteinyl leukotriene type I receptors in rat basophilic leukemia (RBL)-1 cells, large amplitude Ca(2+) oscillations, CRAC channel activity, and downstream Ca(2+)-dependent nuclear factor of activated T cells (NFAT)-driven gene expression are all exclusively maintained by the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule (STIM) 1. However, stimulation of tyrosine kinase-coupled FCεRI receptors evoked Ca(2+) oscillations and NFAT-dependent gene expression through recruitment of both STIM2 and STIM1. We conclude that different agonists activate different STIM proteins to sustain Ca(2+) signals and downstream responses.

Mitofusin 2 regulates STIM1 migration from the Ca2+ store to the plasma membrane in cells with depolarized mitochondria.

Store-operated Ca2+ channels in the plasma membrane (PM) are activated by the depletion of Ca2+ from the endoplasmic reticulum (ER) and constitute a widespread and highly conserved Ca2+ influx pathway. After store emptying, the ER Ca2+ sensor STIM1 forms multimers, which then migrate to ER-PM junctions where they activate the Ca2+ release-activated Ca2+ channel Orai1. Movement of an intracellular protein to such specialized sites where it gates an ion channel is without precedence, but the fundamental question of how STIM1 migrates remains unresolved. Here, we show that trafficking of STIM1 to ER-PM junctions and subsequent Ca2+ release-activated Ca2+ channel activity is impaired following mitochondrial depolarization. We identify the dynamin-related mitochondrial protein mitofusin 2, mutations of which causes the inherited neurodegenerative disease Charcot-Marie-Tooth IIa in humans, as an important component of this mechanism. Our results reveal a molecular mechanism whereby a mitochondrial fusion protein regulates protein trafficking across the endoplasmic reticulum and reveals a homeostatic mechanism whereby mitochondrial depolarization can inhibit store-operated Ca2+ entry, thereby reducing cellular Ca2+ overload.

Mast cell CRAC channel as a novel therapeutic target in allergy.

PURPOSE OF REVIEW: This review describes recent advances in our understanding of a major Ca-entry pathway, the Ca release-activated Ca (CRAC) channel, that is central to mast cell activation. RECENT FINDINGS: Animals in which the genes encoding the CRAC channel have been deleted have severely compromised mast cell function and reduced allergic responses. These functional consequences reflect the ability of CRAC channels to activate a range of spatially and temporally distinct responses in mast cells, which contribute to both rapid and slow phases of an allergic response. In addition, the cells can sustain their own activation through positive feedback cycles that involve CRAC channels. Drugs that inhibit CRAC channels are proving effective in treatment of allergic responses both in vitro and in animal models of asthma. SUMMARY: CRAC channels comprise a new therapeutic target for combating allergies including asthma.

Targeting Ca2+ release-activated Ca2+ channel channels and leukotriene receptors provides a novel combination strategy for treating nasal polyposis.

BACKGROUND: Nasal polyposis is a chronic inflammatory disease of the upper respiratory tract that affects around 2% of the population and almost 67% of patients with aspirin-intolerant asthma. Polyps are rich in mast cells and eosinophils, resulting in high levels of the proinflammatory cysteinyl leukotrienes. OBJECTIVES: To better understand the role of the proinflammatory leukotrienes in nasal polyposis, we asked the following questions: (1) How do nasal polyps produce leukotriene C(4) (LTC(4))? (2) Can LTC(4) feed back in a paracrine way to maintain mast cell activation? (3) Could a combination therapy targeting the elements of this feed-forward loop provide a novel therapy for allergic disease? METHODS: We have used immunohistochemistry, enzyme immunoassay, and cytoplasmic calcium ion (Ca(2+)) imaging to address these questions on cultured and acutely isolated human mast cells from patients with polyposis. RESULTS: Ca(2+) entry through store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels in polyps produced LTC(4) in a manner dependent on protein kinase C. LTC(4) thus generated activated mast cells through cysteinyl leukotriene type I receptors. Hence Ca(2+) influx into mast cells stimulates LTC(4) production, which then acts as a paracrine signal to activate further Ca(2+) influx. A combination of a low concentration of both a CRAC channel blocker and a leukotriene receptor antagonist was as effective at suppressing mast cell activation as a high concentration of either antagonist alone. CONCLUSION: A drug combination directed against CRAC channels and leukotriene receptor antagonist suppresses the feed-forward loop that leads to aberrant mast cell activation. Hence our results identify a new potential strategy for combating polyposis and mast cell-dependent allergies.

CRAC channels and Ca2+ signaling in mast cells.

Mast cells are integral members of the immune system. Upon activation by a rise in cytoplasmic Ca2+, they release a battery of paracrine signals, chemokines, and cytokines, which help sculpt the subsequent immune response. Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane is central for driving most of these responses. The molecular basis of the CRAC channel has been identified, with Orai1 forming the channel pore. Recent work has revealed that a range of mast cell responses are activated by spatially restricted Ca2+ signals just below the plasma membrane. These Ca2+ microdomains can activate cytosolic enzymes, leading to the generation of intracellular messengers as well as proinflammatory molecules like LTC4. In this review, we describe key features of CRAC channels in mast cells, how they generate local Ca2+ signals, and how the cell can decode these restricted signals to generate a raft of responses.

Decoding of cytoplasmic Ca(2+) oscillations through the spatial signature drives gene expression.

Cytoplasmic Ca(2+) oscillations are a universal signaling mode that activates numerous cellular responses [1, 2]. Oscillations are considered the physiological mechanism of Ca(2+) signaling because they occur at low levels of stimulus intensity [3]. Ca(2+) oscillations are proposed to convey information in their amplitude and frequency, leading to activation of specific downstream targets [4-6]. Here, we report that the spatial Ca(2+) gradient within the oscillation is key. Ca(2+) oscillations in mast cells evoked over a range of agonist concentrations in the presence of external Ca(2+) were indistinguishable from those in the absence of Ca(2+) when plasmalemmal Ca(2+) extrusion was suppressed. Nevertheless, only oscillations with accompanying Ca(2+) entry through store-operated CRAC channels triggered gene expression. Increased cytoplasmic Ca(2+) buffering prevented oscillations but not gene activation. Local Ca(2+) influx and not global Ca(2+) oscillations therefore drives gene expression at physiological levels of stimulation. Rather than serving to maintain Ca(2+) oscillations by replenishing stores, we suggest that the role of oscillations might be to activate CRAC channels, thereby ensuring the generation of spatially restricted physiological Ca(2+) signals driving gene activation. Furthermore, we show that the spatial profile of a Ca(2+) oscillation provides a novel mechanism whereby a pleiotropic messenger specifically activates gene expression.

Intercellular Ca2+ wave propagation involving positive feedback between CRAC channels and cysteinyl leukotrienes.

Mast cells are key components of the immune system, where they help orchestrate the inflammatory response. Aberrant mast cell activation is linked to a variety of allergic diseases, including asthma, eczema, rhinitis, and nasal polyposis, which in combination affect up to 20% of the population in industrialized countries. On activation, mast cells release a variety of signals that target the bronchi and vasculature and recruit other immune cells to the inflammatory site. Prominent among such signals are the cysteinyl leukotrienes, a family of potent proinflammatory lipid mediators comprising leukotriene C(4) (LTC(4)), LTD(4), and LTE(4). LTC(4), the parent compound, is secreted from mast cells following Ca(2+) influx through store-operated calcium release-activated calcium (CRAC) channels. Here, we show that activated mast cells release a paracrine signal that evokes Ca(2+) signals in spatially separate resting mast cells. The paracrine signal was identified as a cysteinyl leukotriene because 1) RNAi knockdown or pharmacological block of the 5-lipoxygenase enzyme prevented activated mast cells from stimulating resting cells. 2) Block of cysteinyl leukotriene type I receptors on resting mast cells with the clinically prescribed receptor antagonist montelukast prevented their activation by active mast cells. 3) RNAi knockdown of cysteinyl leukotriene type I receptors on resting cells prevented them from responding to the paracrine signal derived from activated mast cells. 4) Purified LTC(4) evoked Ca(2+) signals in mast cells that were identical to those triggered by the paracrine signal. Low levels of stimulus intensity released sufficient levels of leukotriene to activate resting cells. Leukotriene secretion still occurred tens of minutes after stimulation, suggesting a role as a long-lasting trigger in mast cell activation. Stimulation of the cysteinyl leukotriene receptor activated CRAC channels and evoked prominent store-operated Ca(2+) entry. This resulted in further cysteinyl leukotriene production, triggering a positive feedback cascade. Acutely isolated mast cells from patients with allergic rhinitis exhibited store-operated Ca(2+) influx through CRAC channels and responded to cysteinyl leukotrienes. Histological analysis of samples taken from patients revealed clustering of mast cells, often located within 20 microm of each other, a distance sufficient for paracrine signaling by leukotrienes to operate effectively. We conclude that a positive-feedback cascade involving CRAC channels and cysteinyl leukotrienes constitute a novel mechanism for sustaining mast cell activation.

Sustained activation of the tyrosine kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+ release-activated Ca2+ channels.

Mast cell activation involves cross-linking of IgE receptors followed by phosphorylation of the non-receptor tyrosine kinase Syk. This results in activation of the plasma membrane-bound enzyme phospholipase Cgamma1, which hydrolyzes the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol trisphosphate. Inositol trisphosphate raises cytoplasmic Ca2+ concentration by releasing Ca2+ from intracellular stores. This Ca2+ release phase is accompanied by sustained Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. Here, we find that engagement of IgE receptors activates Syk, and this leads to Ca2+ release from stores followed by Ca2+ influx. The Ca2+ influx phase then sustains Syk activity. The Ca2+ influx pathway activated by these receptors was identified as the CRAC channel, because pharmacological block of the channels with either a low concentration of Gd3+ or exposure to the novel CRAC channel blocker 3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide or RNA interference knockdown of Orai1, which encodes the CRAC channel pore, all prevented the increase in Syk activity triggered by Ca2+ entry. CRAC channels and Syk are spatially close together, because increasing cytoplasmic Ca2+ buffering with the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis failed to prevent activation of Syk by Ca2+ entry. Our results reveal a positive feedback step in mast cell activation where receptor-triggered Syk activation and subsequent Ca2+ release opens CRAC channels, and the ensuing local Ca2+ entry then maintains Syk activity. Ca2+ entry through CRAC channels therefore provides a means whereby the Ca2+ and tyrosine kinase signaling pathways can interact with one another.

Local Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels stimulates production of an intracellular messenger and an intercellular pro-inflammatory signal.

Ca2+ entry through store-operated Ca2+ channels drives the production of the pro-inflammatory molecule leukotriene C4 (LTC4) from mast cells through a pathway involving Ca2+-dependent protein kinase C, mitogen-activated protein kinases ERK1/2, phospholipase A2, and 5-lipoxygenase. Here we examine whether local Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane stimulates this signaling pathway. Manipulating the amplitude and spatial extent of Ca2+ entry by altering chemical and electrical gradients for Ca2+ influx or changing the Ca2+ buffering of the cytoplasm all impacted on protein kinase C and ERK activation, generation of arachidonic acid and LTC4 secretion, with little change in the bulk cytoplasmic Ca2+ rise. Similar bulk cytoplasmic Ca2+ concentrations were achieved when CRAC channels were activated in 0.25 mm external Ca2+ versus 2 mm Ca2+ and 100 nm La3+, an inhibitor of CRAC channels. However, despite similar bulk cytoplasmic Ca2+, protein kinase C activation and LTC4 secretion were larger in 2 mm Ca2+ and La3+ than in 0.25 mm Ca2+, consistent with the central involvement of a subplasmalemmal Ca2+ rise. The nonreceptor tyrosine kinase Syk coupled CRAC channel opening to protein kinase C and ERK activation. Recombinant TRPC3 channels also activated protein kinase C, suggesting that subplasmalemmal Ca2+ rather than a microdomain exclusive to CRAC channels is the trigger. Hence a subplasmalemmal Ca2+ increase in mast cells is highly versatile in that it triggers cytoplasmic responses through generation of intracellular messengers as well as long distance changes through increased secretion of paracrine signals.

All-or-none activation of CRAC channels by agonist elicits graded responses in populations of mast cells.

In nonexcitable cells, receptor stimulation evokes Ca(2+) release from the endoplasmic reticulum stores followed by Ca(2+) influx through store-operated Ca(2+) channels in the plasma membrane. In mast cells, store-operated entry is mediated via Ca(2+) release-activated Ca(2+) (CRAC) channels. In this study, we find that stimulation of muscarinic receptors in cultured mast cells results in Ca(2+)-dependent activation of protein kinase Calpha and the mitogen activated protein kinases ERK1/2 and this is required for the subsequent stimulation of the enzymes Ca(2+)-dependent phospholipase A(2) and 5-lipoxygenase, generating the intracellular messenger arachidonic acid and the proinflammatory intercellular messenger leukotriene C(4). In cell population studies, ERK activation, arachidonic acid release, and leukotriene C(4) secretion were all graded with stimulus intensity. However, at a single cell level, Ca(2+) influx was related to agonist concentration in an essentially all-or-none manner. This paradox of all-or-none CRAC channel activation in single cells with graded responses in cell populations was resolved by the finding that increasing agonist concentration recruited more mast cells but each cell responded by generating all-or-none Ca(2+) influx. These findings were extended to acutely isolated rat peritoneal mast cells where muscarinic or P2Y receptor stimulation evoked all-or-none activation of Ca(2+)entry but graded responses in cell populations. Our results identify a novel way for grading responses to agonists in immune cells and highlight the importance of CRAC channels as a key pharmacological target to control mast cell activation.