Article Figures & Data
Figures
- Fig. 1.
Effect of LPA on ovarian cancer cell adhesion. A, LPA increases cellular adhesion to type I collagen. Cells were cultured for 18 h in the presence (▪, 80 μm) or absence (□) of LPA and analyzed for adhesion to wells coated by passive adsorption with type I collagen. Cells (1.5 × 105) were added to wells for 1 h at 37°C. After washing to remove nonadherent cells, bound cells were fixed and enumerated using an ocular micrometer by counting 10 high-power fields. Data represent the mean and SD from triplicate experiments (∗∗, P < 0.005 relative to control). B, LPA induces expression of β1 integrin. Cells were cultured in the absence (Lane 1) or presence (Lane 2) of LPA as described above. Cells were lysed and lysates (20 μg) were electrophoresed on 9% SDS-polyacrylamide gels and immunoblotted with anti-β1 integrin, followed by peroxidase-conjugated secondary antibody and enhanced chemiluminescence detection. C, LPA enhances collagen-induced proMMP-2 processing. Cells were cultured for 24 h in the absence or presence of LPA (80 μm) before subculturing onto three-dimensional collagen gels (12) . Conditioned media were analyzed for processing of proMMP-2 by gelatin zymography. Lane 1, control cells; Lane 2, LPA-pretreated cells. Arrows, the migration positions of pro- (top) and active-MMP-2 (bottom).
- Fig. 2.
LPA promotes proMMP-2 processing and enhances net MMP activity. A, concentration dependence of LPA-induced proMMP-2 processing. Cells (105) were cultured for 24 h with increasing concentrations of LPA as indicated (μm), and conditioned media were analyzed for activation of proMMP-2 by gelatin zymography. Arrows, the migration positions of pro- (top) and active MMP-2 (bottom). B, LPA-induced proMMP-2 processing is PTX sensitive. Cells were cultured in the absence of LPA (Lanes 1 and 4) or in the presence of LPA (80 μm, Lanes 2 and 3). Addition of PTX (100 ng/ml) abrogated LPA-induced proMMP-2 processing (Lane 3). Control cells (Lane 4) were treated with Con A (20 μg/ml) to induce proMMP-2 activation. Arrows, the migration positions of pro- (top) and active MMP-2 (bottom). C, reverse zymogram of TIMP activity. Cells were cultured in the absence (Lane 1) or presence (Lane 2) of LPA (80 μm) and analyzed for TIMP-1 and -2 expression by reverse zymography on 15% SDS-polyacrylamide gels. Arrows a and b, the migration positions of TIMP-1 and -2 standards, respectively. D, LPA enhances net MMP activity. Cells were treated for 24 h with LPA at the concentrations indicated, and conditioned media (200 μl) were evaluated for MMP activity using the quenched fluorescent substrate (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-Dpa-Ala-Arg (7.5 μm) and by monitoring fluorescence emission at 396 nm using an excitation wavelength of 326 nm (∗∗, P < 0.005 relative to untreated control).
- Fig. 3.
Effect of LPA on MT1-MMP expression, processing, and distribution. A, Western blot of MT1-MMP. Cells (105) were cultured in the absence (Lanes 1–4) or presence (Lanes 5 and 6) of LPA (80 μm) or MMP-I (Lanes 2, 4, and 6), and extracts (20 μg) were analyzed for MT1-MMP processing by electrophoresis on 9% SDS-polyacrylamide gels under reducing conditions and immunoblotting for MT1-MMP followed by peroxidase-conjugated secondary antibody and enhanced chemiluminescence detection. The migration position of molecular weight standards is indicated; numbers on right, Mr in thousands. B, immunofluorescence of MT1-MMP. Cells were cultured in the absence (left) or presence (right) of LPA (80 μm, 24 h) and processed for immunofluorescence microscopy using biotinylated anti-MT1-MMP (1:200) and streptavidin-conjugated FITC as described in “Materials and Methods.”
- Fig. 4.
LPA promotes chemotactic migration. Cells (105) were cultured for 24 h in the presence of LPA at the concentration indicated before seeding onto a porous polycarbonate filter (8-μm pore) coated on the underside with type I collagen (10 μg/ml). After incubation for 4 h to permit migration, nonmigrating cells were removed from the upper chamber, filters were stained, and migrating cells adherent to the underside of the filter were enumerated using an ocular micrometer and counting a minimum of 10 high-powered fields. Data are expressed as relative migration (number of cells/field) and represent the mean and SD of quadruplicate experiments.
- Fig. 5.
LPA promotes haptotactic motility. A, cells (3 × 103) were cultured for 24 h in the absence or presence of LPA (80 μm) or MMPI (10 μm) as indicated before plating on coverslips coated with colloidal gold overlaid with type I collagen (50 μg/ml). Cells were allowed to migrate for 26 h, and phagokinetic tracks were visualized using darkfield illumination. B, semiquantitative analysis of migration area. The area of phagokinetic tracks was evaluated using computer-assisted image analysis and NIHImage. C, untreated controls; LPA, cells treated with 80 μm LPA; Inh, cells treated with MMPI (10 μm); LPA+Inh, cells treated with LPA and MMPI. (∗, P < 0.001 relative to control untreated cells).
- Fig. 6.
LPA enhances in vitro wound closure. A, confluent monolayers of cells were wounded with a uniform scratch, rinsed to remove debris, and incubated in the absence or presence of LPA (80 μm) or MMPI (10 μm) as indicated for 24 h. Photographs indicate relative wound closure as monitored by visual examination using a Zeiss microscope. Fields shown are representative of the width of quadruplicate wounds made in triplicate cultures. B, semiquantitative analysis of wound closure. Relative wound closure was determining by measuring the width of the wounds on photographic images using digital needlepoint calipers. Data are expressed as percentage wound closure, relative to the width of control wounds photographed at time zero (not shown). C, untreated controls; LPA, cells treated with 80 μm LPA; Inh, cells treated with MMPI (10 μm); LPA+Inh, cells treated with LPA and MMPI. (∗, P < 0.001 relative to control untreated cells).
- Fig. 7.
Effect of LPA on invasive activity. Cells (105) were added to porous polycarbonate filters (8-μm pore) coated with Matrigel (11 μg) in the absence or presence of LPA (80 μm) or MMPI (10 μm) as indicated. After incubation for 48 h, conditioned media and filters were collected. A, filters were stained, and invading cells were enumerated using an ocular micrometer to count a minimum of 10 high-powered fields. Data are expressed as relative invasion (number of cells/field) and represent the mean and SD of quadruplicate experiments. (∗∗, P < 0.005 relative to controls in the absence of LPA and MMPI). B, conditioned media were analyzed for MMP activity by gelatin zymography. Arrows, the migration positions of pro- (top) and active MMP-2 (bottom).
Volume 61, Issue 7
