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Mechanism of sphingosine 1-phosphate- and lysophosphatidic acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

Costello RW, Maloney M, Atiyeh M, Gleich G, Walsh MT - Int J Mol Sci (2011)

Bottom Line: IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model.The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells.Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland; E-Mails: rcostello@rcsi.ie (R.W.C.); micmaloney@rcsi.ie (M.M.); matiyeh@rcsi.ie (M.A.).

ABSTRACT
The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

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S1P and LPA induce Gi-mediated ERK phosphorylation and differential up-regulation of ICAM-1 and CCL-26. IMR32 cells in differentiation medium were treated with: S1P (A, C, E) (1 μM) or LPA (1 μM) (B, D, E) for the indicated times. Cells were harvested for cytoplasmic protein and subjected to Western blotting with antibody to phosphor-ERK (A, B, top panels) then ERK2 (A, B, bottom panels) for normalization Blots shown are representative of three similar experiments. Real-time PCR was carried out on cDNA from cells stimulated with S1P or LPA (both 1 μM) in the presence or absence of pre-treatment overnight with the MEK/ERK inhibitor PD98059 (50 μM) or the Gi protein coupling inhibitor, pertussis toxin (PTX) (0.1 μg/mL) (C, D, E), using primers for ICAM-1 (C, D) or CCL-26 (E) or β-actin (C, D, E). Results are expressed as fold increase in ICAM-1/β-actin or CCL-26/β-actin ratio over untreated cells. Mean ± SEM, n = 4; * p < 0.05, LPA-induced fold increase over untreated; † p < 0.05, PD98059 or PTX-induced reduction in LPA-mediated CCL26 up-regulation. Panel F shows that the baseline absolute ratio of ICAM-1 or CCL-26 to β-actin is not significantly different in the presence of either inhibitor pre-treatment.
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f2-ijms-12-03237: S1P and LPA induce Gi-mediated ERK phosphorylation and differential up-regulation of ICAM-1 and CCL-26. IMR32 cells in differentiation medium were treated with: S1P (A, C, E) (1 μM) or LPA (1 μM) (B, D, E) for the indicated times. Cells were harvested for cytoplasmic protein and subjected to Western blotting with antibody to phosphor-ERK (A, B, top panels) then ERK2 (A, B, bottom panels) for normalization Blots shown are representative of three similar experiments. Real-time PCR was carried out on cDNA from cells stimulated with S1P or LPA (both 1 μM) in the presence or absence of pre-treatment overnight with the MEK/ERK inhibitor PD98059 (50 μM) or the Gi protein coupling inhibitor, pertussis toxin (PTX) (0.1 μg/mL) (C, D, E), using primers for ICAM-1 (C, D) or CCL-26 (E) or β-actin (C, D, E). Results are expressed as fold increase in ICAM-1/β-actin or CCL-26/β-actin ratio over untreated cells. Mean ± SEM, n = 4; * p < 0.05, LPA-induced fold increase over untreated; † p < 0.05, PD98059 or PTX-induced reduction in LPA-mediated CCL26 up-regulation. Panel F shows that the baseline absolute ratio of ICAM-1 or CCL-26 to β-actin is not significantly different in the presence of either inhibitor pre-treatment.

Mentions: To determine the intracellular signaling mechanisms that could mediate S1P- and/or LPA-induced up-regulation of ICAM-1 on IMR32 cells, activation of the ERK MAP kinase was examined. Both S1P (Figure 2A) and LPA (Figure 2B) induced ERK phosphorylation but with different time courses. S1P induced activation of ERK was observable at 30 min of stimulation and remained robust at 1–2 h (Figure 2A) while LPA induced a more rapid ERK activation which had fallen to baseline levels between 30 and 60 min of stimulation (Figure 2B). To further determine intracellular mediators involved in S1P- and LPA-induced ICAM-1 up-regulation, IMR32 cells were incubated with S1P (1 μM, 2 h) or LPA (1 μM, 30 min). These treatment times were chosen based on the time course for ICAM-1 induction (Figure 1C) which suggested that these were the optimal times for S1P and LPA respectively. Cells were pre-treated with either the MEK inhibitor PD98059, which prevents ERK phosphorylation or with pertussis toxin (PTX), to inhibit the Gi protein, to which all the receptors S1P1, S1P3 and LPA1–3 can couple. S1P-induced (Figure 2C) or LPA-induced (Figure 2D) up-regulation of ICAM-1 was measured by real-time PCR and compared between cells pre-treated with PD98059 or PTX and non-pre-treated cells. This revealed that up-regulation of ICAM-1 by both S1P and by LPA was dependent on both ERK phosphorylation and on Gi coupling. This is consistent with the recently demonstrated mechanism of S1P-induced up-regulation of ICAM-1 in airway epithelial cells [34].


Mechanism of sphingosine 1-phosphate- and lysophosphatidic acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

Costello RW, Maloney M, Atiyeh M, Gleich G, Walsh MT - Int J Mol Sci (2011)

S1P and LPA induce Gi-mediated ERK phosphorylation and differential up-regulation of ICAM-1 and CCL-26. IMR32 cells in differentiation medium were treated with: S1P (A, C, E) (1 μM) or LPA (1 μM) (B, D, E) for the indicated times. Cells were harvested for cytoplasmic protein and subjected to Western blotting with antibody to phosphor-ERK (A, B, top panels) then ERK2 (A, B, bottom panels) for normalization Blots shown are representative of three similar experiments. Real-time PCR was carried out on cDNA from cells stimulated with S1P or LPA (both 1 μM) in the presence or absence of pre-treatment overnight with the MEK/ERK inhibitor PD98059 (50 μM) or the Gi protein coupling inhibitor, pertussis toxin (PTX) (0.1 μg/mL) (C, D, E), using primers for ICAM-1 (C, D) or CCL-26 (E) or β-actin (C, D, E). Results are expressed as fold increase in ICAM-1/β-actin or CCL-26/β-actin ratio over untreated cells. Mean ± SEM, n = 4; * p < 0.05, LPA-induced fold increase over untreated; † p < 0.05, PD98059 or PTX-induced reduction in LPA-mediated CCL26 up-regulation. Panel F shows that the baseline absolute ratio of ICAM-1 or CCL-26 to β-actin is not significantly different in the presence of either inhibitor pre-treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3116188&req=5

f2-ijms-12-03237: S1P and LPA induce Gi-mediated ERK phosphorylation and differential up-regulation of ICAM-1 and CCL-26. IMR32 cells in differentiation medium were treated with: S1P (A, C, E) (1 μM) or LPA (1 μM) (B, D, E) for the indicated times. Cells were harvested for cytoplasmic protein and subjected to Western blotting with antibody to phosphor-ERK (A, B, top panels) then ERK2 (A, B, bottom panels) for normalization Blots shown are representative of three similar experiments. Real-time PCR was carried out on cDNA from cells stimulated with S1P or LPA (both 1 μM) in the presence or absence of pre-treatment overnight with the MEK/ERK inhibitor PD98059 (50 μM) or the Gi protein coupling inhibitor, pertussis toxin (PTX) (0.1 μg/mL) (C, D, E), using primers for ICAM-1 (C, D) or CCL-26 (E) or β-actin (C, D, E). Results are expressed as fold increase in ICAM-1/β-actin or CCL-26/β-actin ratio over untreated cells. Mean ± SEM, n = 4; * p < 0.05, LPA-induced fold increase over untreated; † p < 0.05, PD98059 or PTX-induced reduction in LPA-mediated CCL26 up-regulation. Panel F shows that the baseline absolute ratio of ICAM-1 or CCL-26 to β-actin is not significantly different in the presence of either inhibitor pre-treatment.
Mentions: To determine the intracellular signaling mechanisms that could mediate S1P- and/or LPA-induced up-regulation of ICAM-1 on IMR32 cells, activation of the ERK MAP kinase was examined. Both S1P (Figure 2A) and LPA (Figure 2B) induced ERK phosphorylation but with different time courses. S1P induced activation of ERK was observable at 30 min of stimulation and remained robust at 1–2 h (Figure 2A) while LPA induced a more rapid ERK activation which had fallen to baseline levels between 30 and 60 min of stimulation (Figure 2B). To further determine intracellular mediators involved in S1P- and LPA-induced ICAM-1 up-regulation, IMR32 cells were incubated with S1P (1 μM, 2 h) or LPA (1 μM, 30 min). These treatment times were chosen based on the time course for ICAM-1 induction (Figure 1C) which suggested that these were the optimal times for S1P and LPA respectively. Cells were pre-treated with either the MEK inhibitor PD98059, which prevents ERK phosphorylation or with pertussis toxin (PTX), to inhibit the Gi protein, to which all the receptors S1P1, S1P3 and LPA1–3 can couple. S1P-induced (Figure 2C) or LPA-induced (Figure 2D) up-regulation of ICAM-1 was measured by real-time PCR and compared between cells pre-treated with PD98059 or PTX and non-pre-treated cells. This revealed that up-regulation of ICAM-1 by both S1P and by LPA was dependent on both ERK phosphorylation and on Gi coupling. This is consistent with the recently demonstrated mechanism of S1P-induced up-regulation of ICAM-1 in airway epithelial cells [34].

Bottom Line: IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model.The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells.Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland; E-Mails: rcostello@rcsi.ie (R.W.C.); micmaloney@rcsi.ie (M.M.); matiyeh@rcsi.ie (M.A.).

ABSTRACT
The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

Show MeSH
Related in: MedlinePlus