H19-7 cells were generously provided by Dr. Kwang C. Chung (Yonsei University, Seoul, Republic of Korea). LPA (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate), 5-bromodeoxyuridine (BrdU), fatty acid-free bovine serum albumin and pertussis toxin (PTX) were purchased from Sigma (St. Louis, MO, USA). Wortmannin and GF109203 were purchased from Tocris Bioscience (Bristol, UK). TOP Flash and FOP Flash luciferase reporters were purchased from Upstate Biotechnology (Lake Placid, NY, USA). Alexa Fluor 568-conjugated Annexin V was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and G418 were obtained from Invitrogen Life Technologies. All other reagents were of analytical grade or the highest purity available.

H19-7 cells, conditionally immortalized, embryonic (day 17) rat hippocampal cells with a temperature-sensitive SV40 large T antigen (16), were maintained at 33ºC (the permissive temperature at which SV40 large T is functional) in DMEM supplemented with 10% FBS, 0.2 mg/ml G418, 50 U/ml penicillin and 50 mg/ml streptomycin. For differentiation, H19-7 cells were shifted to 39ºC to eliminate the expression of the functional large T antigen, then incubated for 24 h in DMEM supplemented with N2 (N2 medium), and treated with LPA. The differentiation of H19-7 cells into neuronal cells was confirmed by the expression of neuronal markers, such as neurofilament-M and neuron specific enolase, and cell morphology as previously described (17). To determine effect of pharmacological inhibitors on the TCF transcriptional activation and inhibitory phosphorylation of GSK3β induced by LPA, the H19-7 cells were pretreated with the inhibitor for 1 h before LPA treatment.

Apoptosis was measured by staining with Annexin V conjugated to Alexa Fluor 568 (Life Technologies, Grand Island, NY, USA) as previously described (2). H19-7 cells were collected, washed with phosphate-buffered saline (PBS) and resuspended in 100 μl binding buffer [10 mM HEPES (pH 7.4), 140 mM NaCl and 2.5 mM CaCl₂]. The cells were then incubated with 5 μl Annexin V-Alexa Fluor 568 for 15 min at room temperature in the dark, followed by the addition of 400 μl binding buffer. The cells were analyzed by flow cytometry using a Guava EasyCyte (GE Healthcare, Piscataway, NJ, USA).

H19-7 cells were collected by brief centrifugation at 500 × g and resuspended in lysis buffer [10 mM HEPES (pH 7.5), 10 mM KCl, 1 mM EGTA, 1 mM EDTA, 1% Nonidet P-40, 1 mM Na₃VO₄, 5 mM NaF and protease inhibitor cocktail]. Following incubation on ice for 10 min, cell lysates were centrifuged at 4,000 × g for 1 min. Supernatants containing cytoplasm were collected and the remaining nuclear pellet was resuspended in lysis buffer containing 0.1% sodium dodecyl sulphate (SDS). The protein concentrations within each lysate were determined by the Bradford assay (Bio-Rad, Richmond, CA, USA). The fractionated extracts were mixed with SDS sample buffer and equal quantities of the samples were loaded and separated by SDS-polyacrylamide gel electrophoresis (8 or 10% reducing gels), transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) and blocked with 5% non-fat milk. The membranes were incubated in primary antibody overnight at 4ºC. Subsequent to this, the membranes were washed in TBST [10 mM Tris, 140 mM NaCl and 0.1% Tween-20 (pH 7.6)], incubated with the appropriate secondary antibody and washed again in TBST. Bands were visualized by enhanced chemiluminescence and exposed to X-ray film.

Differentiating H19-7 cells cultivated on chamber slides (Nalgene, Rochester, NY, USA) were treated with LPA. The cells were fixed and permeabilized as described in a previous study (2), processed for immunocytochemistry with mouse anti-β-tublin III (Sigma) or rabbit anti-β-catenin (Abcam, Cambridge, MA, USA), and subsequently processed with Alexa Fluor 568- or Alexa Fluor 488-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA). The stained cells were observed using a fluorescence microscope (Axio Scope A1; Zeiss, Oberkochen, Germany) with a digital camera attachment.

H19-7 cells were transiently transfected with a luciferase reporter and Renilla luciferase (pRL-TK; Promega Corporation, Madisson, WI, USA) with the indicated plasmids, using FuGene HD (Roche Molecular Biochemicals, Mannheim Germany). The luciferase reporter constructs TOP Flash and FOP Flash (Upstate Biotechnology) were used to determine the TCF/lymphoid enhancing factor (LEF) transcription activity. Following transfection for 6 h, the cells were cultivated in N2 medium for 18 h and to induce differentiation as described previously. The cells were treated with LPA or vehicle for 4 h and collected. The cell extracts were prepared by rinsing each plate twice with PBS and lysing the cells in 150 μl Reporter Assay Lysis buffer (Promega Corporation). The cell lysates were collected, normalized for protein content and assayed using a dual luciferase assay system (Promega Corporation). They were analyzed for firefly luciferase (TOP Flash or FOP Flash) activity, and the reactions were quenched and immediately reanalyzed for Renilla luciferase activity using an Autolumat Luminometer (Berthhold Technologies, Oak Ridge, TN, USA).

Results are presented as the mean ± SEM. Statistical significance was analyzed by one-way analysis of variance followed by a Dunnett’s multiple test or a paired t-test using Prism (GradPad Software, La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

In a previous study, it was demonstrated that LPA stimulated the inactivation of GSK-3 in H19-7 cells through LPA₁ and LPA₂(2). As β-catenin is a substrate for GSK3 and is degraded following phosphorylation by GSK3, and as LPA activates G proteins, such as Gq, Gi/o and G12/13 (via coupling to LPA₁ and LPA₂), LPA may activate β-catenin/TCF signaling in H19-7 cells. To determine whether LPA activated β-catenin/TCF signaling, cytoplasmic and nuclear levels of β-catenin in the H19-7 cells treated with LPA were investigated by immunocytochemistry and western blot analysis. An increase in the level and the nuclear translocation of β-catenin was detected by immunocytochemistry in the H19-7 cells (Fig. 1A). In addition, an increase in the cytoplasmic levels of β-catenin and the subsequent increase in the levels of nuclear β-catenin was demonstrated by western blot analysis (Fig. 1B). An increase in the level of nuclear β-catenin was also detected within 30 min (data not shown). To determine whether LPA activated β-catenin-mediated transcriptional activation, the TCF reporter activity was analyzed in differentiating H19-7 cells transfected with TCF luciferase reporter. TCF with LPA reporter activity was ~2-fold that without LPA, and was similar to that induced by transfection with a plasmid encoding β-catenin truncated N-terminal 90 amino acids (b-CatΔ90) acting as a constitutively active mutant of β-catenin (CA-β-catenin) (Fig. 1C).

Our previous study demonstrated that LPA induced cell survival in a PTX-dependent manner by the post-translational upregulation of Mcl-1 through the inhibitory phosphorylation of GSK3 in differentiating H19-7 cells. The inhibitory phosphorylation of GSK3 is mediated predominantly by PKC and partly by mitogen activated protein kinases, PKA and phosphatidylinositol 3-kinase (PI3K) in the H19-7 cells (2). To determine the mechanism by which LPA stimulates β-catenin/TCF signaling, the effect of pharmacological inhibitors of PKC, PI3K and Gi/o on the LPA-induced activation of the TCF reporter activity and on the inhibitory phosphorylation of GSK3β at Ser9 by LPA was analyzed. GF109203 (a PKC inhibitor) and PTX (a Gi/o specific inhibitor) significantly decreased the LPA-induced activation of TCF reporter activity, whereas wortmannin (a PI3K inhibitor) did not significantly decrease this activation. By contrast, the LPA-induced inhibitory phosphorylation of GSK3β was marginally decreased by PTX and not by wortmannin, while GF109203 completely inhibited the phosphorylation (Fig. 2). The observation that PTX significantly inhibited the LPA-induced β-catenin/TCF signaling, with a marginal decrease in the inhibitory phosphorylation of GSK3β, suggested that LPA may stimulate β-catenin/TCF transactivation via signaling pathways that are independent of the direct inhibition of GSK3.

LPA induced cell survival in a dose-responsive manner in the differentiating H19-7 cells; however, the H19-7 cells treated with LPA were not stained with BrdU, suggesting that LPA induced cell survival by protecting H19-7 cells from cell death, not by inducing cell proliferation (data not shown). To determine whether LPA-mediated β-catenin/TCF signaling was involved in protecting H19-7 cells from cell death, the cells were transfected with a plasmid expressing either CA-β-catenin or empty vector and a plasmid expressing GFP. Transfected cells differentiated and apoptosis was detected by flow cytometry as described in the Materials and methods. Apoptosis was significantly suppressed in the H19-7 cells expressing CA-β-catenin compared with that of the control cells (Fig. 3).