Requirement of Osteopontin in the migration and protection against Taxol-induced apoptosis via the ATX-LPA axis in SGC7901 cells

Abstract

Background: Autotaxin (ATX) possesses lysophospholipase D (lyso PLD) activity, which converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). The ATX-LPA signaling axis has been implicated in angiogenesis, chronic inflammation and tumor progression. Osteopontin (OPN) is an important chemokine involved in the survival, proliferation, migration, invasion and metastasis of gastric cancer cells. The focus of the present study was to investigate the relationship between the ATX-LPA axis and OPN.

Results: In comparison with non-treated cells, we found that the ATX-LPA axis up-regulated OPN expression by 1.92-fold in protein levels and 1.3-fold in mRNA levels. The ATX-LPA axis activates LPA2, Akt, ERK and ELK-1 and also protects SGC7901 cells from apoptosis induced by Taxol treatment.

Conclusions: This study provides the first evidence that expression of OPN induced by ATX-LPA axis is mediated by the activation of Akt and MAPK/ERK pathways through the LPA2 receptor. In addition, OPN is required for the protective effects of ATX-LPA against Taxol-induced apoptosis and ATX-LPA-induced migration of SGC7901 cells.

Figure 1

LPA and ATX/LPC induce OPN expression in SGC7901 cells. (A) Time course of OPN stimulation utilizing ATX (50 ng/ml) + LPC (20 μM) (ATX/LPC2). OPN expression was examined in SGC7901 cells by Western blot and real time-PCR. SGC7901 cells were treated with ATX, LPC, LPA, ATX/LPC1, ATX/LPC2 or DMEM/0.1% BSA. Total protein and RNA was extracted for Western blot (C) and real time -PCR (B), respectively. Figure1 D showed the densitometric ratio of OPN protein/α-tubulin. Statistical analysis was performed using Student's t test. *p < 0.05. The ATX/LPC concentration curve [4] for stimulated motility provides a justification for the concentrations of ATX/LPC chosen throughout the manuscript. LPA or ATX/LPC stimulates OPN production in additional cell lines (eg, SMMC7721 in our study: ATX-LPA axis induces expression of OPN in hepatic cancer cell SMMC7721) as well as in SGC7901 cells.

Figure 2

ATX/LPC-regulated OPN expression is mediated by LPA2 receptor and activation of Akt and MAPK/ERK. SGC7901 cells were exposed to ATX, LPC, LPA, ATX/LPC2 or DMEM/0.1% BSA for 24 hours or 30 min, and then total RNA and protein were extracted for real time-PCR (A-D) and Western blot (E, F), respectively. (E) Immunoblotting was performed to detect the phosphorylation status of ERK and Akt. Total ERK and Akt levels were assessed as control. (F) The effects of the LPA inhibitor, ERK inhibitor and Akt inhibitor on ATX/LPC2-induced OPN expression in SGC7901 cells. SGC7901 cells were treated with Ki16425, PD98059, LY294002 or DMSO (0.1%; a solvent control) for 45 min prior to ATX/LPC2 treatment. 24 hours following stimulations, OPN expression was measured by Western blot analysis. All experiments were performed in triplicate and a representative result is shown here. Figure 2G showed the densitometric ratio of OPN protein/α-tubulin. Statistical analysis was performed using Student's t test. *p < 0.05.

Figure 3

Determination of Elk-1 activities in SGC7901 cells. SGC7901 cells were transfected with either the pFR-Luc plasmid (reporter plasmid), or the pFA2-Elk1 plasmid (fusion trans-activator plasmid). Cells were then treated with ATX/LPC2, in the absence or presence of DMSO, Ki16425, PD98059, LY294002, harvested and the luciferase activity was measured. Statistical analysis was performed using Student's t test. *p < 0.05.

Figure 4

Requirement of OPN in migration induced by ATX-LPA axis protects against Taxol-induced apoptosis in SGC7901 cells. (A) Verification of siRNA knockdown of the OPN protein by Western blot showed a significant reduction of OPN protein in clone3 (SGC7901-siRNA-OPN). (B) The effect of OPN on ATX-LPA-induced migration was assayed using a Transwell assay. Either SGC7901, SGC7901-siRNA-vehicle, or SGC7901-siRNA-OPN cells (1 × 10⁵) were added in the upper chambers of the transwells, incubated in DMEM/0.1% BSA or medium containing ATX, LPC, LPA, ATX/LPC2 and allowed to migrate for 48 hours at 37°C. Migrated cells on the lower chamber were quantified. (D) Flow cytometric analysis of apoptosis: SGC7901, SGC7901-siRNA-vehicle, or SGC7901-siRNA-OPN cells were treated with Taxol (50 nM) in the absence or presence of each group: ATX, LPC, LPA, ATX/LPC2 or DMEM/0.1% BSA for 24 hours. Apoptosis was analyzed by flow cytometry. Data shown in Figure 4C and E represents the mean ± SD from three individual experiments. Data are mean ± SD of triplicate determinations. Statistical analysis was performed using Student's t test. *p < 0.05.