Early LPS-induced ERK activation in retinal pigment epithelium cells is dependent on PIP2-PLC☆

This article presents additional data regarding the study “The phospholipase D pathway mediates the inflammatory response of the retinal pigment epithelium” [1]. The new data presented here show that short exposure of RPE cells to lipopolysaccharide (LPS) induces an early and transient activation of the extracellular signal-regulated kinase (ERK1/2). This early ERK1/2 activation is dependent on phosphatidylinositol bisphosphate-phospholipase C (PIP2-PLC). On the contrary, neither the phospholipase D 1 (PLD1) nor the PLD2 inhibition is able to modulate the early ERK1/2 activation induced by LPS in RPE cells.


Keywords:
Retinal pigment epithelium (RPE) Inflammation Phosphatidylinositol bisphosphatephospholipase C (PIP 2 -PLC) Phospholipase D (PLD) Extracellular signal-regulated kinase (ERK1/2) Lipopolysaccharide (LPS) a b s t r a c t This article presents additional data regarding the study "The phospholipase D pathway mediates the inflammatory response of the retinal pigment epithelium" [1]. The new data presented here show that short exposure of RPE cells to lipopolysaccharide (LPS) induces an early and transient activation of the extracellular signal-regulated kinase (ERK1/2). This early ERK1/2 activation is dependent on phosphatidylinositol bisphosphate-phospholipase C (PIP 2 -PLC). On the contrary, neither the phospholipase D 1 (PLD1) nor the PLD2 inhibition is able to modulate the early ERK1/2 activation induced by LPS in RPE cells. &

Data accessibility
Data is provided within the article

Value of the data
The data can be useful to other scientists investigating the effects of LPS on RPE cells. The data provide additional information regarding the LPS-induced ERK1/2 signaling in RPE cells.
Results shown here demonstrate that the early and the late LPS-induced ERK1/2 activation are differentially modulated by PIP2-PLC and PLD pathways.

Data
The data presented here show that in RPE cells, ERK1/2 activation induced by 5 min treatment with LPS depends on PIP 2 -PLC but is not affected by classical PLDs inhibition.

Statistical analysis
Statistical analysis was performed using ANOVA followed by Bonferroni's test to compare means. p-Values lower than 0.05 were considered statistically significant. Data represent the mean value 7 SD of at least three independent experiments. The WBs shown are a representative image of samples from at least three independent experiments.

Results
As shown in Fig. 1, 5 min exposure to LPS (10 mg/ml) induced a strong activation of ERK1/2 (120% compared to the control condition) in ARPE-19 cells. This activation is transient since it is no longer Fig. 1. ERK1/2 activation in ARPE-19 cells exposed to LPS. ARPE-19 cells were treated with LPS (10 mg/ml) or ultra pure water (control condition) for 5 min or 2 h. ERK1/2 activation was evaluated by WB assays using anti-phospho ERK1/2 (pERK1/2) antibody. Numbers to the right indicate molecular weights and the bar graph shows the densitometry values of pERK1/2/ α-Tubulin expressed as arbitrary units as ratio of the control. Asterisks indicate significant differences with respect to the control condition (**p o 0.01). observed after 2 h treatment with LPS. The transient ERK1/2 activation observed after 5 min stimulation with LPS was only prevented by the PIP 2 -PLC inhibitor U73122 but was not affected by PLD1 or PLD2 inhibition (Fig. 2). These data demonstrate that the LPS-induced early ERK1/2 depends exclusively on PIP 2 -PLC (Fig. 2), while PLD2 activation is necessary to maintain ERK1/2 phosphorylation after 24 h treatment [1].