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Activation of ERK1/2 by store-operated calcium entry in rat parotid acinar cells.

Soltoff SP, Lannon WA - PLoS ONE (2013)

Bottom Line: Agents and conditions that blocked SOCE in native cells, including 2-aminoethyldiphenyl borate (2-APB), SKF96363, and removal of extracellular Ca(2+), also reduced TG- and carbachol-stimulated ERK1/2 phosphorylation.TG-promoted ERK1/2 phosphorylation was blocked when SRC and Protein Kinases C (PKC) were inhibited, and it was blocked in cells pretreated with β-adrenergic agonist isoproterenol.These observations demonstrate that ERK1/2 is activated by a selective mechanism of Ca(2+) entry (SOCE) in these cells, and suggest that ERK1/2 may contribute to events downstream of SOCE.

View Article: PubMed Central - PubMed

Affiliation: Beth Israel Deaconess Medical Center, Department of Medicine, Division of Signal Transduction, Harvard Medical School, Boston, Massachussetts, USA. ssoltoff@bidmc.harvard.edu

ABSTRACT
The regulation of intracellular Ca(2+) concentration ([Ca(2+)]i) plays a critical role in a variety of cellular processes, including transcription, protein activation, vesicle trafficking, and ion movement across epithelial cells. In many cells, the activation of phospholipase C-coupled receptors hydrolyzes membrane phosphoinositides and produces the depletion of endoplasmic reticulum Ca(2+) stores, followed by the sustained elevation of [Ca(2+)]i from Ca(2+) entry across the plasma membrane via store-operated Ca(2+) entry (SOCE). Ca(2+) entry is also increased in a store-independent manner by arachidonate-regulated Ca(2+) (ARC) channels. Using rat parotid salivary gland cells, we examined multiple pathways of Ca(2+) entry/elevation to determine if they activated cell signaling proteins and whether this occurred in a pathway-dependent manner. We observed that SOCE activates extracellular signal-related kinases 1 and 2 (ERK1/2) to ∼3-times basal levels via a receptor-independent mechanism when SOCE was initiated by depleting Ca(2+) stores using the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin (TG). TG-initiated ERK1/2 phosphorylation increased as rapidly as that initiated by the muscarinic receptor agonist carbachol, which promoted an increase to ∼5-times basal levels. Notably, ERK1/2 phosphorylation was not increased by the global elevation of [Ca(2+)]i by Ca(2+) ionophore or by Ca(2+) entry via ARC channels in native cells, although ERK1/2 phosphorylation was increased by Ca(2+) ionophore in Par-C10 and HSY salivary cell lines. Agents and conditions that blocked SOCE in native cells, including 2-aminoethyldiphenyl borate (2-APB), SKF96363, and removal of extracellular Ca(2+), also reduced TG- and carbachol-stimulated ERK1/2 phosphorylation. TG-promoted ERK1/2 phosphorylation was blocked when SRC and Protein Kinases C (PKC) were inhibited, and it was blocked in cells pretreated with β-adrenergic agonist isoproterenol. These observations demonstrate that ERK1/2 is activated by a selective mechanism of Ca(2+) entry (SOCE) in these cells, and suggest that ERK1/2 may contribute to events downstream of SOCE.

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Time course of the effects of [Ca2+]i-elevating and other agents on ERK1/2 phosphorylation in rat parotid acinar cells.A. Comparison of effects of carbachol (10 µM), arachidonic acid (8 µM), TG (1 µM), and ionomycin (1 µM). B. Effect of PGE2 (10 µM). C. Quantitative comparison of multiple agents on ERK1/2 phosphorylation relative to basal. Number of individual experiments is indicated at bottom of the bars. ***p<0.001, **p<0.01, *p<0.05 compared to basal.
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pone-0072881-g001: Time course of the effects of [Ca2+]i-elevating and other agents on ERK1/2 phosphorylation in rat parotid acinar cells.A. Comparison of effects of carbachol (10 µM), arachidonic acid (8 µM), TG (1 µM), and ionomycin (1 µM). B. Effect of PGE2 (10 µM). C. Quantitative comparison of multiple agents on ERK1/2 phosphorylation relative to basal. Number of individual experiments is indicated at bottom of the bars. ***p<0.001, **p<0.01, *p<0.05 compared to basal.

Mentions: SOCE plays a critical role in promoting fluid secretion and saliva formation in salivary gland cells [10] Since changes in ion transport protein activation [32], [33], [34] and changes in [Ca2+]i can initiate or modulate cell signaling events in a variety of cells, we compared the effects of [Ca2+]i elevation by SOCE and other mechanisms on ERK1/2 signaling in rat parotid acinar cells. TG, which inhibits Ca2+ uptake into the endoplasmic reticulum Ca2+ stores and thereby initiates SOCE, produced an increase in ERK1/2 activation (phosphorylation) within 2 min (Figure 1A,C). The muscarinic receptor ligand carbachol (CCh), which increases [Ca2+]i in rat parotid acinar cells by activating G-protein-coupled M3 muscarinic receptors, increased ERK1/2 phosphorylation as rapidly as did TG. Of note, the elevation of [Ca2+]i by exposure of cells to the Ca2+ ionophore ionomycin did not activate ERK1/2. The exposure of cells to arachidonic acid (AA), which increases Ca2+ entry into salivary gland and other cells via ARC channels [14], [27], did not activate ERK1/2. PGE2, a product of arachidonic acid metabolism, also did not produce significant changes in ERK1/2 phosphorylation (Figure 1B,C). These results indicate that increases in [Ca2+]iper se do not activate ERK1/2 in these cells [30], [35], and suggest that Ca2+ entry via SOCE is unique in its activation of ERK1/2.


Activation of ERK1/2 by store-operated calcium entry in rat parotid acinar cells.

Soltoff SP, Lannon WA - PLoS ONE (2013)

Time course of the effects of [Ca2+]i-elevating and other agents on ERK1/2 phosphorylation in rat parotid acinar cells.A. Comparison of effects of carbachol (10 µM), arachidonic acid (8 µM), TG (1 µM), and ionomycin (1 µM). B. Effect of PGE2 (10 µM). C. Quantitative comparison of multiple agents on ERK1/2 phosphorylation relative to basal. Number of individual experiments is indicated at bottom of the bars. ***p<0.001, **p<0.01, *p<0.05 compared to basal.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756958&req=5

pone-0072881-g001: Time course of the effects of [Ca2+]i-elevating and other agents on ERK1/2 phosphorylation in rat parotid acinar cells.A. Comparison of effects of carbachol (10 µM), arachidonic acid (8 µM), TG (1 µM), and ionomycin (1 µM). B. Effect of PGE2 (10 µM). C. Quantitative comparison of multiple agents on ERK1/2 phosphorylation relative to basal. Number of individual experiments is indicated at bottom of the bars. ***p<0.001, **p<0.01, *p<0.05 compared to basal.
Mentions: SOCE plays a critical role in promoting fluid secretion and saliva formation in salivary gland cells [10] Since changes in ion transport protein activation [32], [33], [34] and changes in [Ca2+]i can initiate or modulate cell signaling events in a variety of cells, we compared the effects of [Ca2+]i elevation by SOCE and other mechanisms on ERK1/2 signaling in rat parotid acinar cells. TG, which inhibits Ca2+ uptake into the endoplasmic reticulum Ca2+ stores and thereby initiates SOCE, produced an increase in ERK1/2 activation (phosphorylation) within 2 min (Figure 1A,C). The muscarinic receptor ligand carbachol (CCh), which increases [Ca2+]i in rat parotid acinar cells by activating G-protein-coupled M3 muscarinic receptors, increased ERK1/2 phosphorylation as rapidly as did TG. Of note, the elevation of [Ca2+]i by exposure of cells to the Ca2+ ionophore ionomycin did not activate ERK1/2. The exposure of cells to arachidonic acid (AA), which increases Ca2+ entry into salivary gland and other cells via ARC channels [14], [27], did not activate ERK1/2. PGE2, a product of arachidonic acid metabolism, also did not produce significant changes in ERK1/2 phosphorylation (Figure 1B,C). These results indicate that increases in [Ca2+]iper se do not activate ERK1/2 in these cells [30], [35], and suggest that Ca2+ entry via SOCE is unique in its activation of ERK1/2.

Bottom Line: Agents and conditions that blocked SOCE in native cells, including 2-aminoethyldiphenyl borate (2-APB), SKF96363, and removal of extracellular Ca(2+), also reduced TG- and carbachol-stimulated ERK1/2 phosphorylation.TG-promoted ERK1/2 phosphorylation was blocked when SRC and Protein Kinases C (PKC) were inhibited, and it was blocked in cells pretreated with β-adrenergic agonist isoproterenol.These observations demonstrate that ERK1/2 is activated by a selective mechanism of Ca(2+) entry (SOCE) in these cells, and suggest that ERK1/2 may contribute to events downstream of SOCE.

View Article: PubMed Central - PubMed

Affiliation: Beth Israel Deaconess Medical Center, Department of Medicine, Division of Signal Transduction, Harvard Medical School, Boston, Massachussetts, USA. ssoltoff@bidmc.harvard.edu

ABSTRACT
The regulation of intracellular Ca(2+) concentration ([Ca(2+)]i) plays a critical role in a variety of cellular processes, including transcription, protein activation, vesicle trafficking, and ion movement across epithelial cells. In many cells, the activation of phospholipase C-coupled receptors hydrolyzes membrane phosphoinositides and produces the depletion of endoplasmic reticulum Ca(2+) stores, followed by the sustained elevation of [Ca(2+)]i from Ca(2+) entry across the plasma membrane via store-operated Ca(2+) entry (SOCE). Ca(2+) entry is also increased in a store-independent manner by arachidonate-regulated Ca(2+) (ARC) channels. Using rat parotid salivary gland cells, we examined multiple pathways of Ca(2+) entry/elevation to determine if they activated cell signaling proteins and whether this occurred in a pathway-dependent manner. We observed that SOCE activates extracellular signal-related kinases 1 and 2 (ERK1/2) to ∼3-times basal levels via a receptor-independent mechanism when SOCE was initiated by depleting Ca(2+) stores using the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin (TG). TG-initiated ERK1/2 phosphorylation increased as rapidly as that initiated by the muscarinic receptor agonist carbachol, which promoted an increase to ∼5-times basal levels. Notably, ERK1/2 phosphorylation was not increased by the global elevation of [Ca(2+)]i by Ca(2+) ionophore or by Ca(2+) entry via ARC channels in native cells, although ERK1/2 phosphorylation was increased by Ca(2+) ionophore in Par-C10 and HSY salivary cell lines. Agents and conditions that blocked SOCE in native cells, including 2-aminoethyldiphenyl borate (2-APB), SKF96363, and removal of extracellular Ca(2+), also reduced TG- and carbachol-stimulated ERK1/2 phosphorylation. TG-promoted ERK1/2 phosphorylation was blocked when SRC and Protein Kinases C (PKC) were inhibited, and it was blocked in cells pretreated with β-adrenergic agonist isoproterenol. These observations demonstrate that ERK1/2 is activated by a selective mechanism of Ca(2+) entry (SOCE) in these cells, and suggest that ERK1/2 may contribute to events downstream of SOCE.

Show MeSH
Related in: MedlinePlus