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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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Effect of the SOCE blocker SKF96365 on ERK1/2 activation in rat parotid acinar cells.Cells were exposed to different concentrations of SKF96365 (3–20 µM) for 10 min, and subsequently treated for 2 min with the following: A, TG (1 µM); B, carbachol (10 µM), and C, PMA (100 nM). D. Quantitative analysis of ERK1/2 phosphorylation for conditions shown in Figure 6A,B,C. *p<0.05, **p<0.01, ***p<0.001 compared to basal. #p<0.05, ##p<0.01 as indicated. N = 3–5.
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pone-0072881-g006: Effect of the SOCE blocker SKF96365 on ERK1/2 activation in rat parotid acinar cells.Cells were exposed to different concentrations of SKF96365 (3–20 µM) for 10 min, and subsequently treated for 2 min with the following: A, TG (1 µM); B, carbachol (10 µM), and C, PMA (100 nM). D. Quantitative analysis of ERK1/2 phosphorylation for conditions shown in Figure 6A,B,C. *p<0.05, **p<0.01, ***p<0.001 compared to basal. #p<0.05, ##p<0.01 as indicated. N = 3–5.

Mentions: We examined the effects of several blockers of SOCE to evaluate further the contribution of SOCE to ERK1/2 activation. SKF96365 [36] reduced the activation of ERK1/2 by carbachol in a concentration-dependent manner (Figure 6), and there was a trend that it also reduced the activation by TG. (SKF96365 (20 µM) produced a significant reduction using Student’s t-test, but not when the data were analyzed using ANOVA). Notably, SKF96355 was not effective in blocking the activation of ERK1/2 by the phorbol ester PMA, which directly activates PKC. Since TG and carbachol, but not PMA, promote SOCE, the inhibitory effect of SKF96365 on ERK1/2 is consistent with the involvement of SOCE. SKF96365 blocks TRPC proteins [37], which along with other proteins (Orai1, STIM1) make up the complex that conducts SOCE in salivary gland cells [1], [23].


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

Soltoff SP, Lannon WA - PLoS ONE (2013)

Effect of the SOCE blocker SKF96365 on ERK1/2 activation in rat parotid acinar cells.Cells were exposed to different concentrations of SKF96365 (3–20 µM) for 10 min, and subsequently treated for 2 min with the following: A, TG (1 µM); B, carbachol (10 µM), and C, PMA (100 nM). D. Quantitative analysis of ERK1/2 phosphorylation for conditions shown in Figure 6A,B,C. *p<0.05, **p<0.01, ***p<0.001 compared to basal. #p<0.05, ##p<0.01 as indicated. N = 3–5.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756958&req=5

pone-0072881-g006: Effect of the SOCE blocker SKF96365 on ERK1/2 activation in rat parotid acinar cells.Cells were exposed to different concentrations of SKF96365 (3–20 µM) for 10 min, and subsequently treated for 2 min with the following: A, TG (1 µM); B, carbachol (10 µM), and C, PMA (100 nM). D. Quantitative analysis of ERK1/2 phosphorylation for conditions shown in Figure 6A,B,C. *p<0.05, **p<0.01, ***p<0.001 compared to basal. #p<0.05, ##p<0.01 as indicated. N = 3–5.
Mentions: We examined the effects of several blockers of SOCE to evaluate further the contribution of SOCE to ERK1/2 activation. SKF96365 [36] reduced the activation of ERK1/2 by carbachol in a concentration-dependent manner (Figure 6), and there was a trend that it also reduced the activation by TG. (SKF96365 (20 µM) produced a significant reduction using Student’s t-test, but not when the data were analyzed using ANOVA). Notably, SKF96355 was not effective in blocking the activation of ERK1/2 by the phorbol ester PMA, which directly activates PKC. Since TG and carbachol, but not PMA, promote SOCE, the inhibitory effect of SKF96365 on ERK1/2 is consistent with the involvement of SOCE. SKF96365 blocks TRPC proteins [37], which along with other proteins (Orai1, STIM1) make up the complex that conducts SOCE in salivary gland cells [1], [23].

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