Convergent regulation of neuronal differentiation and Erk and Akt kinases in human neural progenitor cells by lysophosphatidic acid, sphingosine 1-phosphate, and LIF: specific roles for the LPA1 receptor

Abstract

The bioactive lysophospholipids lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) have diverse effects on the developing nervous system and neural progenitors, but the molecular basis for their pleiotropic effects is poorly understood. We previously defined LPA and S1P signaling in proliferating human neural progenitor (hNP) cells, and the current study investigates their role in neuronal differentiation of these cells. Differentiation in the presence of LPA or S1P significantly enhanced cell survival and decreased expression of neuronal markers. Further, the LPA receptor antagonist Ki16425 fully blocked the effects of LPA, and differentiation in the presence of Ki16425 dramatically enhanced neurite length. LPA and S1P robustly activated Erk, but surprisingly both strongly suppressed Akt activation. Ki16425 and pertussis toxin blocked LPA activation of Erk but not LPA inhibition of Akt, suggesting distinct receptor and G-protein subtypes mediate these effects. Finally, we explored cross talk between lysophospholipid signaling and the cytokine leukemia inhibitory factor (LIF). LPA/S1P effects on neuronal differentiation were amplified in the presence of LIF. Similarly, the ability of LPA/S1P to regulate Erk and Akt was impacted by the presence of LIF; LIF enhanced the inhibitory effect of LPA/S1P on Akt phosphorylation, while LIF blunted the activation of Erk by LPA/S1P. Taken together, our results suggest that LPA and S1P enhance survival and inhibit neuronal differentiation of hNP cells, and LPA1 is critical for the effect of LPA. The pleiotropic effects of LPA may reflect differences in receptor subtype expression or cross talk with LIF receptor signaling.

Keywords: Akt; Erk; Ki16425; LIF; LPA1; bFGF; lysophosphatidic acid; neural progenitor; neuronal differentiation; sphingosine 1-phosphate.

Figure 1.

LPA and S1P promote hNP cell survival during differentiation. Cells were differentiated by 14-day bFGF withdrawal as described in Materials and Methods section in the presence or absence of 1 µM LPA, 0.1 µM S1P, or 10 ng/mL LIF. Cell viability and counting assays were carried out on day 14. Data are reported as percentage of vehicle-treated, LIF control wells. (a) Cell viability was assessed using CellTiter-Blue® mitochondrial metabolism reagent as described. Results are representative of three independent experiments. (b) Cell number was determined by counting DAPI-stained nuclei using Cellomics automated image analysis, as described.

Figure 2.

LPA and S1P suppress expression of the neuronal marker βIII-tubulin during in vitro differentiation. Cells were differentiated for 14 days in the absence of bFGF and treated with LPA or S1P as indicated. Cells were fixed, stained, imaged, and analyzed as described in Materials and Methods section. (a) Neuronal profiling algorithm for quantification of βIII-tubulin (Tuj) staining. Upper panels: Undifferentiated hNP cells grown in the presence of bFGF. Lower panels: hN2 cells differentiated by withdrawal of bFGF for 14 days. Left panels: Overlay of DAPI-stained nuclei (blue) and tubulin staining in cell bodies and neurites (green). Middle panels: Nucleus identification algorithm image analysis. Objects outlined in blue were identified as valid nuclei and used for further analysis, while objects outlined in orange were rejected based on algorithm criteria for size, shape, intensity, and image border-intersecting criteria. Right panels: Cell body identification and quantification and neurite identification and measurement algorithm image analysis. Cell bodies were identified based on tubulin staining intensity (shown in grayscale); objects outlined in light blue represent a cell body associated with an identified nucleus. Cells with tubulin staining intensity above a set threshold were scored as positive for tubulin expression. Excluded cell bodies are shown in red. Neurites are identified in green tracing and measured by Cellomics neuronal profiling algorithm. (b) Tubulin expression levels are reported as a percentage of cells expressing tubulin above a set threshold. NP: hNP cells grown in the presence of bFGF. N2: hN2 cells differentiated via bFGF for 2 weeks. (c) Average neurite length of cells treated as indicated.

Figure 3.

LPA and S1P suppress expression of the neuronal marker Map2 during in vitro differentiation. Cells were differentiated and analyzed as described in Materials and Methods section and Figure 2. (a) Neuronal profiling algorithm for quantification of Map2 staining. Upper panels: Undifferentiated hNP cells grown in the presence of bFGF. Lower panels: hN2 cells differentiated by withdrawal of bFGF for 14 days. Left panels: Overlay of DAPI-stained nuclei (blue) and Map2 staining in cell bodies and neurites (green). Middle panels: Nucleus identification algorithm image analysis. Objects outlined in blue were identified as nuclei and used for further analysis, while objects outlined in orange were rejected based on size, shape, intensity, and image border-intersecting criteria. Right panels: Cell body identification and quantification and neurite identification and measurement algorithm image analysis. Cell bodies were identified based on Map2 staining intensity (shown in grayscale); objects outlined in light blue represent a cell body associated with an identified nucleus. Cells with Map2 staining intensity above a set threshold were scored as positive for Map2 expression. Excluded cell bodies are shown in red. Neurites are identified in green tracing and measured by Cellomics neuronal profiling algorithm. (b) Map2 expression levels are reported as a percentage of cells expressing Map2 above a set threshold. NP: hNP cells grown in the presence of bFGF. N2: hN2 cells differentiated via bFGF for 2 weeks.

Figure 4.

LPA1 receptor selective antagonist Ki16425 inhibits LPA effects on differentiation of hNP cells. hNP cells were differentiated for 14 days, in the presence of 1 µM LPA or 10 µM Ki16425 for the final 10 days. Cell survival and protein expression were assessed as described. Cell viability (a) and DAPI-stained nucleus count (b) are reported as % of vehicle-treated, LIF control wells. βIII-tubulin (Tuj) expression (c) and Map2 expression (d) are reported as percent of differentiated cells expressing each marker above a set threshold.

Figure 5.

Ki16425 increases length of neurites in differentiated neurons. hNP cells were differentiated for 14 days, in the presence of 1 µM LPA or 10 µM Ki16425 for the final 10 days. Cells were fixed and stained with βIII-tubulin (Tuj) or Map2 as described, and neurite length was quantified using Cellomics neuronal profiling algorithm. (a) Left panels: Overlay of DAPI-stained nuclei (blue) and tubulin staining (green). Middle panels: Nucleus identification algorithm image analysis. Objects outlined in blue were identified as nuclei and used for further analysis, while objects outlined in orange were rejected based on size, shape, intensity, and image border-intersecting criteria. Right panels: Neurite identification algorithm image analysis. Neurites are identified in green tracing and measured by Cellomics neuronal profiling algorithm. (b) Map2 immunofluorescence staining. (c) The average length of neurites in nhN2 cells differentiated under the indicated conditions as shown.

Figure 6.

LPA and S1P decrease Akt phosphorylation and increase Erk phosphorylation in hNP cells. hNP cells were grown in the absence of bFGF for 24 hr and treated with the indicated compounds in fresh media. (a) Cells were treated with 0.1 µM S1P, 1 µM LPA, or 10 µM Ki16425 for 10 min and then harvested for Western blotting analysis with anti-phospho p42/44 Erk Map kinase antibodies. Expression of GAPDH protein was determined as a housekeeping standard. Band intensities were quantified and Erk phosphorylation levels were normalized to GAPDH levels. (b) Cells were treated with 0.1 µM S1P, 1 µM LPA, or 10 µM Ki16425 for 30 minutes, and then harvested for Western blotting analysis with anti-phospho serine 473 Akt kinase antibodies. Expression of GAPDH protein was determined as a housekeeping standard. Band intensities were quantified and Erk phosphorylation levels were normalized to GAPDH levels. (c) hNP cells were grown in the absence of bFGF for 24 hr and treated with the indicated concentrations of Ki16425 in the absence of exogenous LPA for 10 minutes. Cells were harvested and analyzed by Western blotting analysis with anti-phospho p42/44 Erk Map kinase antibodies. (d) hNP cells were treated in duplicate with 1 µM LPA or 0.1 µM S1P with or without pretreatment with 100 ng/mL pertussis toxin (Ptx) for 12 hr. ERK1/2 phosphorylation was assessed after 10-min LPA/S1P treatment, and Akt phosphorylation was assessed after 30-min LPA/S1P treatment. (e) hNP cells were treated in duplicate with 1 µM LPA or 0.1 µM S1P with or without 30-min pretreatment with 2.5 µM bpV(OHpic) or 100 µM sodium vanadate. ERK1/2 phosphorylation was assessed after 10-min LPA/S1P treatment, and Akt phosphorylation was assessed after 30-min LPA/S1P treatment.

Figure 7.

Costimulation with LIF and bFGF alter the effect of LPA and S1P on Erk and Akt signaling pathways. hNP cells were grown in the absence of bFGF for 24 hr and were then stimulated with 20 ng/mL bFGF, 10 ng/mL LIF, 1 µM LPA, or 0.1 µM S1P as indicated. (a) To assess Erk Map kinase activation, cells were treated for 10 min and then harvested for Western blotting analysis with anti-phospho p42/44 Erk Map kinase antibodies. Expression of GAPDH protein was determined as a housekeeping standard. Band intensities were quantified and Erk phosphorylation levels were normalized to GAPDH levels. (b) To assess Akt activation, cells were treated for 30 min and then harvested for Western blotting analysis with anti-phospho serine 473 Akt kinase antibodies. Expression of GAPDH protein was determined as a housekeeping standard. Band intensities were quantified and Erk phosphorylation levels were normalized to GAPDH levels. Right panels: The ability of LPA to regulate Erk and Akt activation levels was compared when added to cells alone or in the presence of bFGF, LIF, or LIF and bFGF. Under each condition, the percent change in phosphorylation in LPA-treated cells versus vehicle controls was determined. *p < .05.

Figure 8.

LPA and S1P inhibit mRNA expression of neuronal markers. hNP cells were differentiated by bFGF withdrawal for 2 weeks treated with LPA, S1P, or LIF, and qPCR analysis was conducted as described in Materials and Methods section. Data are reported as fold change in expression compared with hNP cells grown in bFGF containing media (NP). (a) βIII-tubulin mRNA expression. (b) Map2 mRNA expression. (c) HuC mRNA expression. (d) HuD mRNA expression. (e) ChAT mRNA expression. *p < .05. **p < .01. ***p < .001.