Sphingosine 1-Phosphate Receptor 5 (S1P5) Deficiency Promotes Proliferation and Immortalization of Mouse Embryonic Fibroblasts

Abstract

Sphingosine 1-phosphate (S1P), a bioactive lipid, interacts with five widely expressed G protein-coupled receptors (S1P1-5), regulating a variety of downstream signaling pathways with overlapping but also opposing functions. To date, data regarding the role of S1P5 in cell proliferation are ambiguous, and its role in controlling the growth of untransformed cells remains to be fully elucidated. In this study, we examined the effects of S1P5 deficiency on mouse embryonic fibroblasts (MEFs). Our results indicate that lack of S1P5 expression profoundly affects cell morphology and proliferation. First, S1P5 deficiency reduces cellular senescence and promotes MEF immortalization. Second, it decreases cell size and leads to cell elongation, which is accompanied by decreased cell spreading and migration. Third, it increases proliferation rate, a phenotype rescued by the reintroduction of exogenous S1P5. Mechanistically, S1P5 promotes the activation of FAK, controlling cell spreading and adhesion while the anti-proliferative function of the S1P/S1P5 signaling is associated with reduced nuclear accumulation of activated ERK. Our results suggest that S1P5 opposes the growth-promoting function of S1P1-3 through spatial control of ERK activation and provides new insights into the anti-proliferative function of S1P5.

Keywords: ERK; FAK; MEF immortalization; S1P5; cell migration; cell proliferation; cell spreading; sphingosine-1phosphate.

Figure 1

Primary S1P5^−/− MEFs are resistant to cellular senescence and prone to immortalization. (A), population doublings of S1P5^+/+ and S1P5^−/− primary MEFs over serial passaging according to 3T3 protocol. S1P5^+/+ and S1P5^−/− MEFs were each isolated from three different embryos. (B), wild-type and S1P5-deficient MEFs at passage six (P6) and passage 12 (P12), were labeled with BrdU for 3 h. The incorporated BrdU was visualized by immunofluorescence. DAPI staining was used to visualize nuclei. Data represent means ± SEM of three different clones performed in triplicates (*, p < 0.0114, ***, p < 0.0007). (C), colony formation capacity of S1P5^+/+ and S1P5^−/− MEFs at passage 6 (P6). Wild-type and S1P5-deficient MEFs were plated in a 6 well-plate and 10 days later cells were fixed and stained with crystal violet to visualize colony formation. Representative images are shown in the left panel. Right panel: means of colony number ± SEM of three different clones performed in triplicates (***, p < 0.0001). (D), S1P5^+/+ and S1P5^−/− MEFs at passage 6 (P6) and passage 12 (P12), were stained for SA-β-Gal activity. SA-β-Gal-positive cells were counted in more than five fields, and results represent the means ± SEM of three different clones performed in triplicates (***, p < 0.0001).

Figure 2

Morphological characterization of S1P5^+/+ and S1P5^−/− immortalized clones. (A), phenotype of immortalized MEFs. Representative phase contrast images from low confluence cultures of S1P5^+/+ and S1P5 ^−/− cells at passage 18 after fixation and staining with crystal violet are shown in the left panel. Right panel: quantification of the ratio of maximal length (L) versus cell width (W) from n = 30 cells per condition from four independent experiments performed between passages 18 to 22. Each dot represents a cell (***, p < 0.0001). (B), cell area and actin organization in S1P5^−/− cells. S1P5^+/+ and S1P5^−/− 2 MEFs were plated on coverslips and 24 h later were fixed and stained for F-Actin (Phalloidin-red) and DNA (Hoechst 33,342-blue). The left panel shows representative images. Scale bar, 10 μm. The right panel shows the quantification of the cell area in µm² based on F-Actin staining. The mean surface area was calculated using ImageJ software. The data are represented in a dot plot, where each point represents one cell and the black lines indicate means ± SEM (***, p < 0.0001; S1P5^+/+, 2612 ± 255, n = 41; S1P5^−/− 1467 ± 147, n = 50).

Figure 3

Impaired adhesion, spreading and migration in S1P5^−/− cells. S1P5^+/+ and S1P5^−/− MEFs were plated in non-coated (A) or fibronectin-coated (B) 24-well plates and incubated for 0.5, 1, and 2 h. Cells were washed and the adherent cells were stained with crystal violet, and the staining intensity was quantified by spectrophotometry at 595 nm. The results represent fold cell adhesion (of wild-type cells) and are means ± SEM of three independent experiments (**, p < 0.0083; *, p < 0.0149; **, p < 0.0073; *, p < 0.0226). (C), cell spreading and actin organization in S1P5^−/− cells. S1P5^+/+ and S1P5^−/− 2 MEFs were plated on coverslips for the indicated times, fixed and stained for F-Actin (Phalloidin-red) and DNA (Hoechst 33,342-blue). The upper panel shows representative images. Scale bar: 10 μm. The lower panel shows the quantification of the cell area (in µm²) based on F-Actin staining. The mean surface area from 100 individual cells was calculated using the ImageJ software. The data are shown as dot plot, where each point represents one cell and the black lines indicate means ± SEM (***, p < 0.0001). (D), equal numbers of S1P5^+/+ and S1P5^−/− MEF cells were seeded in 8-µm pore Transwell cell culture inserts and incubated for 8 h. The cells that migrated to the bottom of the well were fixed and stained with crystal violet. The left panel shows representative images and the right panel the relative migration (expressed as percentage of control cells). The means ± SEM from four experiments is shown (***, p < 0.0001). (E), impaired FAK phosphorylation in S1P5^−/− MEFs. S1P5^+/+ and S1P5^−/− cells were serum-starved for 4 h and then treated with S1P (1 µM) for the indicated times or grown in medium containing 10% FBS for 24 h (right panel). Total cell extracts were analyzed by SDS/PAGE with antibodies against phosphorylated FAK, FAK and tubulin.

Figure 4

Growth properties of immortalized S1P5-deficient cells. (A), S1P5^+/+ and S1P5 ^−/− cells were plated at equal cell number and the cell number was counted daily over four days. Results are representative from four independent experiments performed in triplicates from MEFs at passages 18 to 22. (B), cell survival of S1P5^+/+ and S1P5^−/− MEFs (passages 18–22) was assessed by MTT assays at the indicated times after seeding. Results are representative from three independent experiments performed in triplicates. (C), colony formation capacity of immortalized S1P5^+/+ and S1P5^−/− MEFs between passages 18–22. Low number of cells were plated and allowed to grow for 10 days. Cells were then fixed and stained with crystal violet to visualize colony formation. Graph shows the means of colony number ± SEM from three independent experiments performed in triplicates (*, p < 0.0357; **, p < 0.0017). (D), cell death/viability was evaluated by trypan blue staining. S1P5^+/+ and S1P5^−/− cells were plated at equal cell number and the number of trypan blue-positive cells was counted at the indicated time points. Results are means ± SEM from three independent experiments performed in triplicates. (ns, not significant). (E), equal number of S1P5^+/+, S1P5^+/+ clone 2 (S1P5^+/+ 2) and S1P5^−/− 2 cells were plated and the cell number was counted 72 h later. Results represent means of the relative number of cells ± SEM from three independent experiments performed in triplicates (***, p < 0.0007; ns, p = 0.6279). (F), S1P5 rescues the S1P5^−/− phenotype. Immortalized MEFs S1P5^−/− clone 1 and clone 2 were transiently transfected with empty vector (GFP) or a vector containing mS1P5-GFP. The total cell number was evaluated at different post transfection times. Results represent means of the relative number of cells ± SEM of three independent experiments performed in triplicates (**, p < 0.0058; **, p < 0.0099; **, p < 0.0079; ns, p = 0.1879).

Figure 5

S1P5 induces growth arrest. (A), representative images showing CHO cells stably expressing GFP or mS1P5-GFP and stained for DNA with DAPI. Scale bar: 10 μm. Right panel shows GFP and mS1P5-GFP levels in CHO cells analyzed by Western blot using antibodies against GFP. (B), pharmacological characterization of mS1P5R-GFP. Agonist stimulation of CHO cells ectopically expressing mS1P5-GFP or GFP alone. Membrane fractions were isolated and stimulated with increased concentrations of S1P or FTY720-P. The graph is representative of two independent experiments performed in duplicate. (C), growth properties of CHO cells stably expressing mS1P5R-GFP or GFP. CHO mS1P5-GFP or CHO GFP cells were plated at equal cell number and the cell number was counted daily over a period of four days. Growth curves show means ± SEM from three independent experiments performed in triplicates (**, p < 0.0021; *, p < 0.0134; *, p < 0.0157).

Figure 6

S1P5-induced growth inhibition is ligand-dependent. (A), CHO cells expressing mS1P5-GFP or GFP alone were plated at 20,000 cells/well in 12 well-plates. After 16 h, cells were left untreated (NT) or treated with Z-VAD (10 µM), vehicle (DMSO), or FTY720-P (1 µM), and the cell number was counted 48 h post treatment. Graphs show means ± SEM from three independent experiments performed in triplicate (***, p <0.0007; **, p < 0.0022; **, p < 0.0045; ns, p = 0.6309). (B), mS1P5-GFP or GFP expressing CHO cells were plated as in (A) and treated with control antibodies (α-Ctl) or antibodies against S1P (α-S1P). Cells were counted 48 h post treatment. Graphs show means ± SEM from four independent experiments performed in triplicates (*** p < 0.0007; ** p < 0.0048; ns, p = 0.8298). (C), CHO cells expressing mS1P5-GFP or GFP alone were plated as in (A). After 16 h, cells were washed and treated in DMEM supplemented with delipidated FBS (csFBS) for 24 h in the absence or in the presence of S1P (1 µM) or S1P and α-S1P. Cells grown in medium containing 10% FBS were also counted (FBS). Results show cell number 48 h post treatment and are means ± SEM from at least three independent experiments performed in triplicate (***, p < 0.0001; ns, p = 0.6309; **, p < 0.0033; ns, p = 0.6870).

Figure 7

Knockdown of S1P5 promotes cells growth in a S1P dependent manner. (A), S1P5^+/+ and S1P5^−/− cells were plated at 20,000 cells/well and 16 h later, cells were treated with vehicle (DMSO), Z-VAD (10 µM), FTY720-P (1 µM) or VPC23019 (10 µM) + JTE-013 (10 µM). Results show relative cell number expressed as a percentage of control non-treated cells ± SEM from more than three independent experiments performed in triplicates (***, p < 0.0004; ***, p < 0.0007; ***, p < 0.0006; ns, p = 0.8656; **, p < 0.0049; *, p < 0.0240; *, p < 0.0217). (B), S1P5^+/+ and S1P5^−/− cells cultured as in (A) and were either grown in DMEM containing delipidated FBS (csFBS) or in complete medium containing FBS in the presence of control antibodies (α-Ctl) or antibodies against S1P (α-S1P). Cells were counted 48 h post treatment. Results show the relative cell number expressed as a percentage of control non-treated cells ± SEM from three independent experiments performed in triplicate (***, p < 0.0001; ns, p = 0.4685; **, p < 0.0031; ns, p = 0.7922; ***, p < 0.0005).

Figure 8

S1P5 reduces nuclear P-ERK localization. (A), S1P5^+/+ and S1P5^−/− cells were grown in medium containing 10% FBS for 24 h. Total cell extracts were analyzed by SDS/PAGE with antibodies against the indicated proteins. (B), S1P5^+/+ and S1P5^−/− MEFs were cultured as in (A), fixed and stained for phospho-ERK (a-ERK-green), DNA (Hoechst 33,342-blue) and actin (phalloidin-red). Representative images are shown in the upper panel. Scale bar: 10 μm. (C), quantification of nuclear phospho-ERK staining based on anti-ERK and Hoechst 33,342 staining. Images were taken with a 20× objective and the same exposure settings were used for all fluorescence quantifications. Results are expressed as fluorescence intensity per µm² (***, p < 0.0001; S1P5^+/+, 423.3 ± 33.30, n = 52; S1P5^−/− 603.6 ± 30.77, n = 61).

Figure 9

Model of S1P5 function in MEF cell proliferation, spreading and migration. Extracellular S1P (i) decreases FBS-induced ERK activation and subcellular localization via S1P5, (ii) promotes cell proliferation via S1P1-3, and (iii) induces FAK phosphorylation, resulting in cell adhesion, spreading, and migration.