Early LPS-induced ERK activation in retinal pigment epithelium cells is dependent on PIP 2 -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.

Specifications table

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.

1. Data

The data presented here show that in RPE cells, ERK1/2 activation induced by 5 min treatment with LPS depends on PIP2-PLC but is not affected by classical PLDs inhibition.

2. Experimental design, materials and methods

2.1. Retinal-pigmented epithelium cell culture and treatments

Human retinal-pigmented epithelium cells (ARPE-19) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Natocor, Argentina), 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B at 37 ˚C under 5% CO2. Confluent 35 mm diameter cell dishes were serum-starved for 2 h prior to stimulation for 5 min or 2 h with 10 μg/ml of Pseudomonas aeruginosa LPS in serum-free DMEM. Sterile ultra pure water was added to the control condition. In order to inhibit PIP2-PLC and PLDs pathways ARPE-19 cells were preincubated with selective inhibitors for 1 h at 37 °C prior to cell stimulation with LPS. 0.15 μM EVJ was used to inhibit PLD1 activity and 0.5 μM APV to inhibit PLD2. For PIP2-PLC inhibition, cells were preincubated with U73122 (10 μM). DMSO (vehicle of the inhibitors) was added to all conditions to achieve a final concentration of 0.025% [1], [2].

2.2. Western blot analysis (WBs)

After experimental treatment the medium was removed, cells were washed three times with PBS and scraped off with 80 μl ice-cold RIPA lyses buffer (10 mM Tris–HCl (pH 7.4), 15 mM NaCl, 1% Triton X-100, 5 mM NaF, 1 mM Na2VO4 and the complete protease inhibitor cocktail). Protein content of cellular lysates was determined by Bradford method [3] (Bio-Rad Life Science group, #500-0006). Samples were denatured with Laemmli sample buffer at 100 °C for 5 min [4]. Equivalent amounts of proteins (30 μg) were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) and WBs were performed as previously described [1]. Rabbit polyclonal antibody anti-phospho-ERK1/2 (#9101) was from Cell Signaling (Beverly, MA, USA). Mouse monoclonal anti-α Tubulin (DM1-A) (CP06) was from EMD/Biosciences-Calbiochem (San Diego, CA, USA). HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Densitometry values of the immunoreactive bands were determined using ImageJ 1.38 software.

2.3. 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±SD of at least three independent experiments. The WBs shown are a representative image of samples from at least three independent experiments.

3. Results

As shown in Fig. 1, 5 min exposure to LPS (10 µg/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 observed after 2 h treatment with LPS. The transient ERK1/2 activation observed after 5 min stimulation with LPS was only prevented by the PIP2-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 PIP2-PLC (Fig. 2), while PLD2 activation is necessary to maintain ERK1/2 phosphorylation after 24 h treatment [1].

Fig. 1

ERK1/2 activation in ARPE-19 cells exposed to LPS. ARPE-19 cells were treated with LPS (10 µg/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<0.01).

Fig. 2

Role of PIP2-PLC and PLDs in ERK1/2 activation. ARPE-19 cells were preincubated with 0.025% DMSO (control and vehicle conditions), 0.15 µM EVJ, 0.5 µM APV or 10 µM U73122 for 1 h before LPS treatment. Cells were treated with 10 µg/ml LPS or ultra pure water (control condition) for 5 min. 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. (a and b) Indicate significant differences among columns (p<0.05).

Subject area	Biochemistry
More specific subject area	Cell biology
Type of data	WB images, bar graphs
How data was acquired	Western blot. Densitometry values were obtained using the ImageJ software
Data format	Raw and analyzed
Experimental factors	ARPE-19 cells were exposed to LPS. Pharmacologycal inhibitors of PLD1, PLD2 and PIP2-PLC were used.
Experimental features	ERK1/2 activation was evaluated by Western blot
Data source location	Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB), Universidad Nacional del Sur (UNS) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), 8000 Bahía Blanca, Argentina.
Data accessibility	Data is provided within the article