Interaction of monocarboxylate transporter 4 with beta1-integrin and its role in cell migration

Abstract

Monocarboxylate transporter (MCT) 4 is a heteromeric proton-coupled lactate transporter that is noncovalently linked to the extracellular matrix metalloproteinase inducer CD147 and is typically expressed in glycolytic tissues. There is increasing evidence to suggest that ion transporters are part of macromolecular complexes involved in regulating beta(1)-integrin adhesion and cell movement. In the present study we examined whether MCTs play a role in cell migration through their interaction with beta(1)-integrin. Using reciprocal coimmunoprecipitation assays, we found that beta(1)-integrin selectively associated with MCT4 in ARPE-19 and MDCK cells, two epithelial cell lines that express both MCT1 and MCT4. In polarized monolayers of ARPE-19 cells, MCT4 and beta(1)-integrin colocalized to the basolateral membrane, while both proteins were found in the leading edge lamellapodia of migrating cells. In scratch-wound assays, MCT4 knockdown slowed migration and increased focal adhesion size. In contrast, silencing MCT1 did not alter the rate of cell migration or focal adhesion size. Taken together, our findings suggest that the specific interaction of MCT4 with beta(1)-integrin may regulate cell migration through modulation of focal adhesions.

Fig. 1.

Monocarboxylate transporter (MCT) 4 associates with β₁-integrin in ARPE-19 cells. A: immunofluorescence microscopy of frozen sections of ARPE-19 cells labeled with MCT4 and β₁-integrin antibodies. Bar = 10 μm. DIC, differential interference contrast. B and C: the association of MCT1 and MCT4 with β₁-integrin was studied by reciprocal coimmunoprecipitation of ARPE-19 lysates and immunoblotting. B: ARPE-19 detergent-soluble lysates were immunoprecipitated (IP) with β₁-integrin antibody and blotted with MCT4, MCT1, α₃- and α₅-integrin antibodies. Relative amounts of MCT1 and MCT4 coimmunoprecipitated with β₁-integrin were expressed as a percentage of the input fraction ± SE (bottom) C: ARPE-19 lysates were immunoprecipitated with MCT4 and MCT1 antibodies and probed with β₁-integrin antibody. Levels of association were determined as a percentage of the input fraction ± SE (bottom). Ten micrograms of total protein were loaded in “input” lanes; 20 μl of pellet fraction were loaded in “IP” and “control” lanes. Control, beads + lysate.

Fig. 2.

Silencing MCTs/CD147 in ARPE-19 cells does not impact integrin expression. A: RT-PCR was performed on cells to determine the effectiveness of small interfering RNA (siRNA)-mediated silencing of MCT1, MCT4, or CD147 and if there were changes in expression of nontarget mRNAs. Cells were harvested for total RNA preparation 48 h posttransfection. PCR results show that MCT1, MCT4, and CD147 siRNAs efficiently silenced their respective targets. No change in mRNA levels of β₁-integrin was observed. GAPDH and β-actin served as loading controls. B: cells were harvested for protein analysis 72 h posttransfection. Silencing MCT1 and MCT4 resulted in decreased expression of CD147, while silencing of CD147 resulted in decreased expression of MCT1 and MCT4. β₁-Integrin expression was not affected by silencing MCT1, MCT4, or CD147. β-Actin was used as a loading control. Ten micrograms of total protein were loaded per lane.

Fig. 3.

Silencing MCT4 slows cell migration. ARPE-19 cells were wounded using a 1-ml micropipette tip 48 h following transfection with MCT1, MCT4, or CD147 siRNAs. Wounds were measured at 0 h and 25 h postwounding. Top: silencing MCT4 reduced wound closure to 48% as compared with mock-transfected control (69% closure). Silencing CD147 had a similar effect on wound closure (56% closure). Loss of MCT1 expression did not alter cell migration (72% closure), as compared with mock-transfected controls. Bars represent SE. **P < 0.0001 as determined by one-way ANOVA. Bottom: images depicting wounds at 0 h and 25 h postwounding following transfection with MCT1, MCT4, or CD147 siRNAs. Mock, untransfected control; KD, knockdown.

Fig. 4.

MCT4 colocalizes with β₁-integrin at the leading edge of migrating ARPE-19 cells. A: wounded ARPE-19 cell monolayers were fixed 24 h postwounding and immunolabeled with MCT4 and β₁-integrin antibodies. Immunoconfocal micrographs show that MCT4 (red) and β₁-integrin (green) colocalized (yellow, see merge) at the lateral cell borders and in the leading edge of migrating cells. B: moderate levels of MCT1 (red) colocalized with β₁-integrin (green) at lateral cell borders (yellow, see merge) and at the leading edge of wounded cells (see merge and inset). C and D: silencing of MCT4 (C) or MCT1 (D) did not alter the distribution of β₁-integrin (green) to the leading edge. Smaller panels in A–D denote higher magnification of boxed area in larger image. Arrows denote cell-cell borders and arrowheads denote the leading edge. Bar = 10 μm.

Fig. 5.

Focal adhesions are increased in size in MCT4 siRNA-transfected cells. ARPE-19 cell cultures were fixed and labeled with paxillin antibody 24 h after wounding. Shown are immunoconfocal micrographs of mock-transfected cells (A and B), MCT1 siRNA-transfected cells (C and D), and MCT4 siRNA-transfected cells (E and F). Note that when MCT4 was silenced there was an increase in size of the adhesion plaques. Arrows denote large focal adhesions and arrowheads denote small focal adhesions. B, D, and F, higher-magnification images of the boxed region shown in A, C, and E. Bar = 10 μm.