HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6016fjev1 20/13/2381    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chang, W.-C. Articles by Parekh, A. B. Search for Related Content PubMed PubMed Citation Articles by Chang, W.-C. Articles by Parekh, A. B.

Ca²⁺ influx through CRAC channels activates cytosolic phospholipase A₂, leukotriene C₄ secretion, and expression of c-fos through ERK-dependent and -independent pathways in mast cells

Department of Physiology University of Oxford, Oxford, England, UK

¹Correspondence: Department of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT UK. E-mail: anant.parekh{at}physiol.ox.ac.uk

SPECIFIC AIM

The aim of the present work was to investigate mechanisms in a mast cell line, regulating the production of the potent proinflammatory signal leukotriene C₄, a molecule that has been linked to a variety of cardiovascular disorders and asthma. A rise in intracellular Ca²⁺ concentration activates cytosolic phospholipase A₂ (cPLA₂) liberating arachidonic acid, which is then metabolized by the 5-lipoxygenase enzyme to form leukotriene C₄. How these processes are co-ordinated is unknown.

PRINCIPAL FINDINGS

The sarco/endoplasmic reticulum CaATPase inhibitor thapsigargin depleted intracellular Ca²⁺ stores, which resulted in Ca²⁺ release followed by opening of store-operated CRAC channels in the plasma membrane in rat basophilic leukemia (RBL-1) cells, a mast cell line. Ca²⁺ influx, but not Ca²⁺ release, activated the mitogen-activated protein kinases ERK1/2 (Fig. 1 Aa) and robust ERK activation was apparent by 4 min of stimulation with thapsigargin. The CRAC channel blocker 2-APB prevented Ca²⁺ influx from activating ERK (Fig. 1Ab ). U0126, an inhibitor of the upstream mitogen-activated protein kinases MEK1/2, which are needed for ERK activation, prevented Ca²⁺ influx through CRAC channels from activating ERK (Fig. 1Aa ). Mitochondrial depolarization, which impairs CRAC channel activity, also reduced the extent of activation of ERK by thapsigargin (Fig. 1Ac ).

cPLA₂ is activated following phosphorylation on Ser-505 by ERK1/2. Ca²⁺ influx through CRAC channels resulted in significant phosphorylation of cPLA₂ at Ser-505, and this was prevented by suppressing ERK activation with U0126 or the structurally distinct MEK inhibitor PD98059 (Fig. 1B ). Moreover, these inhibitors suppressed arachidonic acid release (Fig. 1C ) and subsequent secretion of leukotriene C₄ following thapsigargin-evoked Ca²⁺ influx (Fig. 1D ). Inhibition of ERK did not impair the cytoplasmic Ca²⁺ rise following stimulation with thapsigargin (Fig. 1E ) or Ca²⁺ flux through CRAC channels (Fig. 1F ). Stimulation of the cells with a physiological trigger (antigen) activated ERK, and this was needed for LTC₄ production.

Ca²⁺ influx through CRAC channels activated ERK indirectly, via recruitment of Ca²⁺-dependent protein kinase C isozymes and ßbeta;1. Ca²⁺ influx triggered, within minutes, translocation of protein kinase C and ßbeta;1 to the plasma membrane, and down-regulation of these enzymes following chronic exposure to phorbol ester prevented Ca²⁺ entry from activating cPLA₂ or secretion of leukotriene C₄. Acute inhibition of Ca²⁺-dependent protein kinase C isozymes with G0–6976 prevented thapsigargin stimulation from activating ERK, stimulating cPLA₂ or promoting leukotriene C₄ secretion. Direct stimulation of protein kinase C with phorbol ester activated ERK, but, in the absence of a cytoplasmic Ca²⁺ rise, this failed to increase cPLA₂ activity. However, cytoplasmic Ca²⁺ and protein kinase C interacted synergistically such that protein kinase C increased the ability of modest Ca²⁺ influx to generate arachidonic acid and leukotriene secretion.

Arachidonic acid is a promiscuous signal that can activate a wide range of intracellular processes. However, we found that ERK tightly correlated the production of arachidonic acid with leukotriene secretion. Ca²⁺ entry through CRAC channels, in addition to activating cPLA₂, stimulated translocation of the 5-lipoxygenase enzyme to the nuclear membrane. This is a critical early step in 5-lipoxygenase activation. Importantly, translocation of 5-lipoxygenase was prevented by U0126. Hence, ERK is a versatile transducer of CRAC channel activity, translating the ensuing Ca²⁺ entry into the co-ordinated activation of two interdependent enzymes.

ERK can trigger gene activation by regulating transcription factors. We found that Ca²⁺ entry through CRAC channels, following just 4 min stimulation with thapsigargin, resulted in increased transcription (Fig. 2 A) and translation (Fig. 2B ) of the immediate early gene c-fos. Paradoxically, neither U0126 (Fig. 2C ) nor PD98059 (Fig. 2D ) significantly impaired c-fos expression, despite blocking cPLA₂ activity and 5-lipoxygenase translocation. Hence, Ca²⁺ influx through CRAC channels induces short-term effects (within minutes), culminating in the generation of important intra- and intercellular messengers as well as longer-term events involving gene expression. The short-term processes (cPLA₂ and 5-lipoxygenase activation) are mediated through ERK activation, whereas the longer term effects are evoked via an ERK-independent pathway that links Ca²⁺ entry to nuclear events (Fig. 3 ).

CONCLUSIONS AND SIGNIFICANCE

Although store-operated CRAC channels are believed to be important physiologically, little solid evidence documents their involvement in any defined process. We show here for the first time that CRAC channels are central to the generation of both intracellular and intercellular signals as well as activation of c-fos gene transcription. We show crosstalk between two key intracellular signaling pathways (calcium and the MAP kinases ERK1/2) in that Ca²⁺ influx activates ERK via protein kinase C and ßbeta;I, and this is central for stimulation of two important enzymes: cytosolic phospholipase A₂ and 5-lipoxygenase. Mitochondria are central regulators of this signaling cascade. Paradoxically, c-fos expression is not dependent on ERK. Ca²⁺ influx through CRAC channels can therefore activate different signaling pathways at the same time, culminating in a range of temporally diverse responses. Finally, our findings identify CRAC channels as an attractive therapeutic target for curbing excessive generation of LTC₄, which is linked to asthma and atherosclerosis.

View larger version (28K): [in this window] [in a new window]   Figure 1. Ca2+ entry through CRAC channels activates cPLA2 and LTC4 secretion through the ERK pathway. Aa) Western blots show that stimulation with 2–5 µM thapsigargin (4 min) results in the phosphorylation of ERK1/2 (denoted in upper panel as ERK1/2-P), indicative of ERK activation. Phosphorylation is prevented by removing external Ca2+. The last lane shows that the MEK1/2 inhibitor U0126 (20 µM pretreatment for 15 min) suppresses thapsigargin-induced ERK phosphorylation. Total ERK2 levels are shown (lower panel), as an indicator of constant loading of protein into each lane. Ab) Western blot showing thapsigargin-evoked ERK activation is prevented by the CRAC channel blocker 2-APB (30 µM). Loading control is shown in lower panel. Ac) Mitochondrial depolarization with either FCCP (5 µM) or rotenone (1 µg/ml) reduces ERK phosphorylation (both applied in the presence of 1 µg/ml oligomycin). Oligomycin alone had only a weak effect. Loading control is shown in the lower panel. B) Thapsigargin stimulates phosphorylation of cPLA2 on Ser-505. Upper panel shows a Western blot with a monoclonal antibody (mAb) that specifically recognizes Ser-505 cPLA2. The lower panel plots quantitative analysis from three such gels. Thapsigargin induced a significant increase in Ser-505 phosphorylation compared with control (P<0.01), and the increase was substantially reduced by either U0126 or PD98059 (P<0.01 for both cases). C) U0126 and PD98059 inhibit arachidonic acid release following CRAC channel opening. D) U0126 inhibits LTC4 secretion in a concentration-dependent manner. E) Cytoplasmic Ca2+ signals arising from store-operated Ca2+ entry are unaffected by U0126 (20 µM; 15 min pretreatment; 33 cells for control and 37 cells for U0126). Cells were pretreated with thapsigargin in Ca2+-free solution for 10 min before external Ca2+ was readmitted. F) The amplitude of ICRAC was not affected by U0126 (5 cells for each condition). *Denotes P < 0.01.

View larger version (18K): [in this window] [in a new window]   Figure 2. Expression of c-fos following Ca2+ entry through CRAC channels. A) RT-polymerase chain reaction (RT-PCR) showing thapsigargin stimulation (4 min) in the presence of external Ca2+ results in c-fos gene expression but not in the absence of external Ca2+ or in the presence of 2-APB. Cells were stimulated with thapsigargin for 4 min and then perfused with Ca2+-free solution for 41 min prior to RNA extraction The upper panel shows control ßbeta;-actin loading. B) Western blotting of nuclear membrane shows that Ca2+ entry promotes c-fos protein expression, and this is suppressed by 2-APB (30 µM; 5 min pretreatment). Same stimulation protocol as in (A). C, upper panel) Depicts a typical nuclear membrane blot probing for c-fos expression in nonstimulated cells and after stimulation with thapsigargin in the absence and then presence of U0126. Aggregate data from four gels are summarized in the histogram. D) As in (C) but now in the presence of PD98059.

View larger version (47K): [in this window] [in a new window]   Figure 3. Ca2+ entry through CRAC channels activates protein kinase C and ßbeta;I, which then activate the MEK/ERK cascade. ERK, in turn, stimulates both cPLA2 and 5-lipoxygenase activities, generating intra-and intercellular messengers within minutes. Ca2+ influx through CRAC channels also activates gene transcription, operating over tens of minutes and occurring through an ERK-independent pathway.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6016fje

This article has been cited by other articles:

				J. W. Putney and G. S. Bird Cytoplasmic calcium oscillations and store-operated calcium influx J. Physiol., July 1, 2008; 586(13): 3055 - 3059. [Abstract] [Full Text] [PDF]

				W.-C. Chang, J. Di Capite, K. Singaravelu, C. Nelson, V. Halse, and A. B. Parekh Local Ca2+ Influx through Ca2+ Release-activated Ca2+ (CRAC) Channels Stimulates Production of an Intracellular Messenger and an Intercellular Pro-inflammatory Signal J. Biol. Chem., February 22, 2008; 283(8): 4622 - 4631. [Abstract] [Full Text] [PDF]

				E. Abad, G. Lorente, N. Gavara, M. Morales, A. Gual, and X. Gasull Activation of Store-Operated Ca2+ Channels in Trabecular Meshwork Cells Invest. Ophthalmol. Vis. Sci., February 1, 2008; 49(2): 677 - 686. [Abstract] [Full Text] [PDF]

				W.-C. Chang, J. Di Capite, C. Nelson, and A. B. Parekh All-or-None Activation of CRAC Channels by Agonist Elicits Graded Responses in Populations of Mast Cells J. Immunol., October 15, 2007; 179(8): 5255 - 5263. [Abstract] [Full Text] [PDF]

				Y. Taketomi, K. Sunaga, S. Tanaka, M. Nakamura, S. Arata, T. Okuda, T.-C. Moon, H.-W. Chang, Y. Sugimoto, K. Kokame, et al. Impaired Mast Cell Maturation and Degranulation and Attenuated Allergic Responses in Ndrg1-Deficient Mice J. Immunol., June 1, 2007; 178(11): 7042 - 7053. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6016fjev1 20/13/2381    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chang, W.-C. Articles by Parekh, A. B. Search for Related Content PubMed PubMed Citation Articles by Chang, W.-C. Articles by Parekh, A. B.