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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 receptors in IMR-32 cells and S1P- or LPA- induced ICAM-1 transcription. IMR32 cells were plated in 6-well tissue culture dishes in differentiation medium (5 × 105 per well) then harvested for: (A) RNA and cDNA preparation and semi-quantitative or quantitative PCR using primers for (A) S1P1–5 or LPA1–3 or (B) total protein preparation and Western blotting for receptors S1P1, S1P3 or LPA1–3. (C) IMR32 cells in differentiation medium were treated with: S1P (1 μM), LPA (1 μM) or TNF (10 ng/mL) for the indicated times, then harvested for RNA and cDNA preparation and real-time PCR using primers for ICAM-1 or β-actin. Results are expressed as fold increase in ICAM-1/β-actin ratio over non-EPO treated (0) cells (set to unity). Mean ± SEM; * p < 0.05, **p < 0.01, significantly increased versus untreated cells.
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f1-ijms-12-03237: S1P and LPA receptors in IMR-32 cells and S1P- or LPA- induced ICAM-1 transcription. IMR32 cells were plated in 6-well tissue culture dishes in differentiation medium (5 × 105 per well) then harvested for: (A) RNA and cDNA preparation and semi-quantitative or quantitative PCR using primers for (A) S1P1–5 or LPA1–3 or (B) total protein preparation and Western blotting for receptors S1P1, S1P3 or LPA1–3. (C) IMR32 cells in differentiation medium were treated with: S1P (1 μM), LPA (1 μM) or TNF (10 ng/mL) for the indicated times, then harvested for RNA and cDNA preparation and real-time PCR using primers for ICAM-1 or β-actin. Results are expressed as fold increase in ICAM-1/β-actin ratio over non-EPO treated (0) cells (set to unity). Mean ± SEM; * p < 0.05, **p < 0.01, significantly increased versus untreated cells.

Mentions: Expression of S1P receptors S1P1 and S1P3 (Figure 1A) and of LPA1–3 (Figure 1B) was confirmed in IMR32 cells maintained in proliferation or differentiation medium by real-time PCR. No expression of S1P2, S1P4 or S1P5 was detected after 40 cycles of PCR in any cells (Figure 1A); as a positive control, expression of all three of these receptors was readily detected on neutrophils (Figure 1A). All further experiments were in differentiating IMR32 cells, as these cells display a strongly cholinergic phenotype in differentiation medium [8,24,38]. We confirmed by Western blotting that, identically to the cDNA expression profile, differentiating IMR32 cells expressed S1P1, S1P3 (Figure 1B) and LPA1–3 proteins (Figure 1B), but not S1P2, S1P4 or S1P5. We determined by real time PCR that both S1P (Figure 1C) and LPA (Figure 1C) increased expression of ICAM-1 two- to three-fold over baseline levels, a similar order of magnitude as the positive control stimulus, TNF- (Figure 1C). However, up-regulation of ICAM-1 expression was induced more transiently by LPA (Figure 1C), compared to S1P which induced a sustained up-regulation between 30 min to 24 h (Figure 1C), similar to TNF- (Figure 1C).


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 receptors in IMR-32 cells and S1P- or LPA- induced ICAM-1 transcription. IMR32 cells were plated in 6-well tissue culture dishes in differentiation medium (5 × 105 per well) then harvested for: (A) RNA and cDNA preparation and semi-quantitative or quantitative PCR using primers for (A) S1P1–5 or LPA1–3 or (B) total protein preparation and Western blotting for receptors S1P1, S1P3 or LPA1–3. (C) IMR32 cells in differentiation medium were treated with: S1P (1 μM), LPA (1 μM) or TNF (10 ng/mL) for the indicated times, then harvested for RNA and cDNA preparation and real-time PCR using primers for ICAM-1 or β-actin. Results are expressed as fold increase in ICAM-1/β-actin ratio over non-EPO treated (0) cells (set to unity). Mean ± SEM; * p < 0.05, **p < 0.01, significantly increased versus untreated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3116188&req=5

f1-ijms-12-03237: S1P and LPA receptors in IMR-32 cells and S1P- or LPA- induced ICAM-1 transcription. IMR32 cells were plated in 6-well tissue culture dishes in differentiation medium (5 × 105 per well) then harvested for: (A) RNA and cDNA preparation and semi-quantitative or quantitative PCR using primers for (A) S1P1–5 or LPA1–3 or (B) total protein preparation and Western blotting for receptors S1P1, S1P3 or LPA1–3. (C) IMR32 cells in differentiation medium were treated with: S1P (1 μM), LPA (1 μM) or TNF (10 ng/mL) for the indicated times, then harvested for RNA and cDNA preparation and real-time PCR using primers for ICAM-1 or β-actin. Results are expressed as fold increase in ICAM-1/β-actin ratio over non-EPO treated (0) cells (set to unity). Mean ± SEM; * p < 0.05, **p < 0.01, significantly increased versus untreated cells.
Mentions: Expression of S1P receptors S1P1 and S1P3 (Figure 1A) and of LPA1–3 (Figure 1B) was confirmed in IMR32 cells maintained in proliferation or differentiation medium by real-time PCR. No expression of S1P2, S1P4 or S1P5 was detected after 40 cycles of PCR in any cells (Figure 1A); as a positive control, expression of all three of these receptors was readily detected on neutrophils (Figure 1A). All further experiments were in differentiating IMR32 cells, as these cells display a strongly cholinergic phenotype in differentiation medium [8,24,38]. We confirmed by Western blotting that, identically to the cDNA expression profile, differentiating IMR32 cells expressed S1P1, S1P3 (Figure 1B) and LPA1–3 proteins (Figure 1B), but not S1P2, S1P4 or S1P5. We determined by real time PCR that both S1P (Figure 1C) and LPA (Figure 1C) increased expression of ICAM-1 two- to three-fold over baseline levels, a similar order of magnitude as the positive control stimulus, TNF- (Figure 1C). However, up-regulation of ICAM-1 expression was induced more transiently by LPA (Figure 1C), compared to S1P which induced a sustained up-regulation between 30 min to 24 h (Figure 1C), similar to TNF- (Figure 1C).

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