Integrin-mediated cell surface recruitment of autotaxin promotes persistent directional cell migration

Abstract

Autotaxin (ATX) is a secreted lysophospholipase D (lysoPLD) that binds to integrin adhesion receptors. We dissected the roles of integrin binding and lysoPLD activity in stimulation of human breast cancer and mouse aortic vascular smooth muscle cell migration by ATX. We compared effects of wild-type human ATX, catalytically inactive ATX, an integrin binding-defective ATX variant with wild-type lysoPLD activity, the isolated ATX integrin binding N-terminal domain, and a potent ATX selective lysoPLD inhibitor on cell migration using transwell and single-cell tracking assays. Stimulation of transwell migration was reduced (18 or 27% of control, respectively) but not ablated by inactivation of integrin binding or inhibition of lysoPLD activity. The N-terminal domain increased transwell migration (30% of control). ATX lysoPLD activity and integrin binding were necessary for a 3.8-fold increase in the fraction of migrating breast cancer cell step velocities >0.7 μm/min. ATX increased the persistent directionality of single-cell migration 2-fold. This effect was lysoPLD activity independent and recapitulated by the integrin binding N-terminal domain. Integrin binding enables uptake and intracellular sequestration of ATX, which redistributes to the front of migrating cells. ATX binding to integrins and lysoPLD activity therefore cooperate to promote rapid persistent directional cell migration.

Keywords: cell adhesion; cell motility; lysophosphatidic acid; lysophospholipase.

Figure 1.

An ATX variant containing an engineered disulfide bond linking the SMB1 and PDE domains retains near-wild-type catalytic activity but does not bind to β3 integrins. A) Schematic diagram of wild-type ATX and the ATX variants and fragments used in these studies. B) Rat ATX structure identifying residues mutated to introduce the SMB1-PDE domain-linking disulfide residue. Note that V279 in the rat sequence corresponds to S279 in human ATX, which was used for our studies. C, D) Comparison of nucleotide phosphodiesterase (C) and lysoPLD (D) activities of wild-type (wt) ATX and ATXLock (means of duplicate determinations). E, F) Binding of β3-integrin-overexpressing CHO (CHO-β3; E) or MDA-MB-231 (F) cells to wild-type ATX, ATX SMB1,2, ATXLock, and fibrinogen (Fn) and BSA as positive and negative controls, respectively, determined in buffer containing either 10 μM Ca²⁺ and Mg²⁺, 500 μM Mn²⁺ or 500 μM Mn²⁺, and 20 μM echistatin, as described in Materials and Methods. Data are means ± sd of triplicate determinations from experiments repeated ≥3 times. Statistical significance evaluated by Student's t test. N.S. not significant (P>0.05). *P < 0.001.

Figure 2.

Integrin binding is necessary for lysoPLD-dependent and -independent effects of ATX on MDA-MB-231 cell migration in transwell assays. A) Effects of increasing concentrations of ATX on MDA-MB-231 cell migration. B) Effects of increasing concentrations of the ATX lysoPLD inhibitor PF8380 on MDA-MB-231 cell transwell migration (open circles) and ATX lysoPLD activity (solid circles). C) Comparison of effects of 1 μg/ml ATX and 1 μg/ml ATXLock on MDA-MB-231 cell migration in the presence or absence of vehicle or 1 μM PF8380. D) Comparison of effects of 1 μg/ml ATX and 1 μg/ml ATXLock on mAVSMC migration in the presence of vehicle or 1 μM PF8380. E) Comparison of effects of 1 μg/ml ATX, 1 μg/ml ATXT201A, and 1 μg/ml SMB1,2 on MDA-MB-231 cell and mAVSMC migration. F) Effects of increasing concentrations of ATX SMB1,2 on MDA-MB-231 cell migration. The number of cells migrating under the indicated experimental conditions was normalized to the number of cells migrating in response to 1 μg/ml ATX. Data are means ± sd of triplicate determinations from experiments repeated ≥3 times. Statistical significance evaluated by Student's t test. N.S., not significant (P>0.05). *P < 0.001.

Figure 3.

ATX stimulates migration of single MDA-MB-231 cells. A, B) Trajectories of 16 randomly selected MDA-MB-231 cells measured by monitoring the position of the cell nucleus every 10 min for 6 h in medium containing 0.1% fatty acid-free BSA with (B) and without (A) 1 μg/ml ATX. C) Individual MDA-MB-231 cells were tracked for 6 h by measuring the position of the cell nucleus every 10 min. Histogram shows the frequency distribution of step velocities measured for 37 tracked steps of the indicated number of MDA-MB-231 cells, determined in the presence of either BSA as control, 1 μg/ml ATX, 1 μg/ml ATX combined with 1 μM PF8380, or the ATX SMB1,2 domain. D) Effect of BSA, ATX, ATX in the presence of 1 μM PF8380, the ATX SMB1,2 domain (see C for number of cells analyzed), 100 ng/ml EGF (100 cells), or 1 μM 18:1 LPA (100 cells) on migration step velocities of individual MDA-MB-231 cells. E) Effect of BSA, ATX, ATX in the presence of 1 μM PF8380, the ATX SMB1,2 domain (see C for number of cells analyzed), 100 ng/ml EGF (100 cells), or 1 μM 18:1 LPA (100 cells) on the fraction of step velocities > 0.7 μm/min. D) Data were analyzed by generalized linear mixed modeling. E) Data were analyzed by 1-way ANOVA (see Materials and Methods for details). Results of pairwise comparisons are indicated using capital italic letters; 2 groups not sharing a letter are significantly different (P<0.05). Box plots show rectangles with 25th, 50th, and 75th percentiles and whiskers extending to the 10th and 90th percentiles.

Figure 4.

ATX increases the directional persistence of MDA-MD-231 cell migration. A) Illustration of data from a model single-cell tracking experiment showing the parameters measured to determine the total, net, and greatest distance moved and directionality. B–F) Effect of BSA, ATX, ATX in the presence of 1 μM PF8380, the ATX SMB1,2 domain (see Fig. 3C for number of cells analyzed), 100 ng/ml EGF (100 cells), or 1 μM 18:0 LPA (100 cells) on the mean migration velocity (B), total distance migrated (C), greatest distance migrated (D), net distance migrated (E), and directional persistence (F) of single MDA-MB-231 cell migration. Data were analyzed by 1-way ANOVA after log transformation. Results of pairwise comparisons are indicated using capital italic letters; 2 groups not sharing a letter are significantly different (P<0.05). Box plots show rectangles with 25th, 50th, and 75th percentiles and whiskers extending to 10th and 90th percentiles.

Figure 5.

Uptake, intracellular accumulation, and redistribution of exogenous ATX by MDA-MB-231 cells. A–C) Time (A), ATX-concentration (B), and temperature dependence (C) of accumulation of Alexa Fluor 488-labeled ATX by MDA-MB-231 cells (means± sd of triplicate determinations from a representative experiment). D) Intracellular localization of Alexa Fluor 488-labeled ATX (green). E) Visualization of intracellular Alexa Fluor 488-labeled ATX (green) and the focal adhesion marker paxilin (red). F) Association of Alex Fluor 488-ATX with MDA-MB-231 cells is inhibited by preincubation with the β3-integrin-blocking antibody 7E3. G) Intracellular localization of Alexa Fluor 488-labeled ATX SMB1,2 domain (green). Cells are counterstained with DAPI (blue) to visualize the nucleus. H–K) Still images from Supplemental Movie S1 show Alexa Fluor 488 localization (green) in a single migrating MDA-MB-231 as it changes direction, at 0 min (H), 3 min (I), 4 min (J), and 5 min (K). Arrows indicate direction of cell migration at indicated times.