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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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Contribution of extracellular Ca2+ to ERK1/2 activation by TG and carbachol in rat parotid acinar cells.A. Cells were suspended in the absence or presence (1.8 mM) of Ca2+, and exposed to TG (1 µM) or carbachol (10 µM) for 2 min. B. Quantitative analysis of ERK1/2 phosphorylation in conditions shown in Figure 4A. ***p<0.001 compared to basal, #p<0.05 as indicated. C. Time course of ERK1/2 phosphorylation in cells exposed to TG (1 µM) in the absence of Ca2+. D. Comparison of ERK1/2 phosphorylation in cells in Ca2+-free conditions and exposed to TG (1 µM, 15 min) or exposed to TG (1 µM, 15 min) followed by Ca2+ (1 mM, 2 min) to initiate SOCE. E. Quantitative analysis of ERK1/2 phosphorylation for conditions shown in Figure 4C and 4D. **p<0.01, ***p<0.001 compared to basal; ###p<0.001 as indicated. N = 3–16.
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pone-0072881-g004: Contribution of extracellular Ca2+ to ERK1/2 activation by TG and carbachol in rat parotid acinar cells.A. Cells were suspended in the absence or presence (1.8 mM) of Ca2+, and exposed to TG (1 µM) or carbachol (10 µM) for 2 min. B. Quantitative analysis of ERK1/2 phosphorylation in conditions shown in Figure 4A. ***p<0.001 compared to basal, #p<0.05 as indicated. C. Time course of ERK1/2 phosphorylation in cells exposed to TG (1 µM) in the absence of Ca2+. D. Comparison of ERK1/2 phosphorylation in cells in Ca2+-free conditions and exposed to TG (1 µM, 15 min) or exposed to TG (1 µM, 15 min) followed by Ca2+ (1 mM, 2 min) to initiate SOCE. E. Quantitative analysis of ERK1/2 phosphorylation for conditions shown in Figure 4C and 4D. **p<0.01, ***p<0.001 compared to basal; ###p<0.001 as indicated. N = 3–16.

Mentions: To determine whether TG-initiated ERK1/2 activation was dependent on Ca2+ entry, we performed experiments in the absence of extracellular Ca2+ to block increases in [Ca2+]i via SOCE. TG promoted small but significant increases in ERK1/2 phosphorylation in cells suspended in the absence of extracellular Ca2+, but TG produced larger increases in the presence of Ca2+ (Figure 4A,B). The activation of ERK1/2 by carbachol also displayed a dependence on extracellular Ca2+ (Figure 4A, B). We examined this more closely and found that in the absence of Ca2+, TG produced a larger increase in ERK1/2 phosphorylation after 2 min than at later times up to 15 min of exposure (Figure 4C,E). Notably, there was a large SOCE-dependent increase in ERK1/2 phosphorylation upon the addition of Ca2+ for 2 min to cells previously exposed to TG for 15 min in Ca2+-free solution (Figure 4D,E).


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

Soltoff SP, Lannon WA - PLoS ONE (2013)

Contribution of extracellular Ca2+ to ERK1/2 activation by TG and carbachol in rat parotid acinar cells.A. Cells were suspended in the absence or presence (1.8 mM) of Ca2+, and exposed to TG (1 µM) or carbachol (10 µM) for 2 min. B. Quantitative analysis of ERK1/2 phosphorylation in conditions shown in Figure 4A. ***p<0.001 compared to basal, #p<0.05 as indicated. C. Time course of ERK1/2 phosphorylation in cells exposed to TG (1 µM) in the absence of Ca2+. D. Comparison of ERK1/2 phosphorylation in cells in Ca2+-free conditions and exposed to TG (1 µM, 15 min) or exposed to TG (1 µM, 15 min) followed by Ca2+ (1 mM, 2 min) to initiate SOCE. E. Quantitative analysis of ERK1/2 phosphorylation for conditions shown in Figure 4C and 4D. **p<0.01, ***p<0.001 compared to basal; ###p<0.001 as indicated. N = 3–16.
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getmorefigures.php?uid=PMC3756958&req=5

pone-0072881-g004: Contribution of extracellular Ca2+ to ERK1/2 activation by TG and carbachol in rat parotid acinar cells.A. Cells were suspended in the absence or presence (1.8 mM) of Ca2+, and exposed to TG (1 µM) or carbachol (10 µM) for 2 min. B. Quantitative analysis of ERK1/2 phosphorylation in conditions shown in Figure 4A. ***p<0.001 compared to basal, #p<0.05 as indicated. C. Time course of ERK1/2 phosphorylation in cells exposed to TG (1 µM) in the absence of Ca2+. D. Comparison of ERK1/2 phosphorylation in cells in Ca2+-free conditions and exposed to TG (1 µM, 15 min) or exposed to TG (1 µM, 15 min) followed by Ca2+ (1 mM, 2 min) to initiate SOCE. E. Quantitative analysis of ERK1/2 phosphorylation for conditions shown in Figure 4C and 4D. **p<0.01, ***p<0.001 compared to basal; ###p<0.001 as indicated. N = 3–16.
Mentions: To determine whether TG-initiated ERK1/2 activation was dependent on Ca2+ entry, we performed experiments in the absence of extracellular Ca2+ to block increases in [Ca2+]i via SOCE. TG promoted small but significant increases in ERK1/2 phosphorylation in cells suspended in the absence of extracellular Ca2+, but TG produced larger increases in the presence of Ca2+ (Figure 4A,B). The activation of ERK1/2 by carbachol also displayed a dependence on extracellular Ca2+ (Figure 4A, B). We examined this more closely and found that in the absence of Ca2+, TG produced a larger increase in ERK1/2 phosphorylation after 2 min than at later times up to 15 min of exposure (Figure 4C,E). Notably, there was a large SOCE-dependent increase in ERK1/2 phosphorylation upon the addition of Ca2+ for 2 min to cells previously exposed to TG for 15 min in Ca2+-free solution (Figure 4D,E).

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