Korean J Physiol Pharmacol.  2016 Sep;20(5):515-523. 10.4196/kjpp.2016.20.5.515.

Molecular association of CD98, CD29, and CD147 critically mediates monocytic U937 cell adhesion

• Kim MY
• Cho JY

• KMID: 2350508
• DOI: http://doi.org/10.4196/kjpp.2016.20.5.515

Abstract

Adhesion events of monocytes represent an important step in inflammatory responses induced by chemokines. The β1-integrin CD29 is a major adhesion molecule regulating leukocyte migration and extravasation. Although several adhesion molecules have been known as regulators of CD29, the molecular interactions between CD29 and its regulatory adhesion molecules (such as CD98 and CD147) have not been fully elucidated. Therefore, in this study, we examined whether these molecules are functionally, biochemically, and cell-biologically associated using monocytic U937 cells treated with aggregation-stimulating and blocking antibodies, as well as enzyme inhibitors. The surface levels of CD29, CD98, and CD147 (but not CD43, CD44, and CD82) were increased. The activation of CD29, CD98, and CD147 by ligation of them with aggregation-activating antibodies triggered the induction of cell-cell adhesion, and sensitivity to various enzyme inhibitors and aggregation-blocking antibodies was similar for CD29-, CD98-, and CD147-induced U937 cell aggregation. Molecular association between these molecules and the actin cytoskeleton was confirmed by confocal microscopy and immunoprecipitation. These results strongly suggest that CD29 might be modulated by its biochemical and cellular regulators, including CD98 and CD147, via the actin cytoskeleton.

Keyword

Actin cytoskeleton; Adhesion molecule; CD29; CD98; CD147; U937 cells

MeSH Terms

Actin Cytoskeleton
Antibodies
Antibodies, Blocking
Chemokines
Enzyme Inhibitors
Immunoprecipitation
Leukocytes
Ligation
Microscopy, Confocal
Monocytes
U937 Cells*
Antibodies
Antibodies, Blocking
Chemokines
Enzyme Inhibitors

Figure

• 

  Fig. 1 Flow cytometric analysis of surface adhesion molecule expression in U937 cells.Surface adhesion molecules [CD29 (β1-integrins), CD43, CD98, and CD147] in U937 cells (1×105) were analyzed by flow cytometry. All staining antibodies were used at 1~5 µg/ml. Mean fluorescence intensity (MFI, percent of control) was calculated using WIN-MDI software from a minimum of 7,500 cells. The results (mean±SD, n=4) show one representative experiment out of three.

• 

  Fig. 2 Cell-cell adhesion induced by ligation of surface adhesion molecules with aggregation-activating antibodies.(A) U937 cells were incubated with pro-aggregative (aggregation-activating) antibodies (1 µg/ml each as IgG1) to CD98 (AHN-18, 1 µg/ml), CD29 (MEM101A, 1 µg/ml), CD147 (M6-1D4, 10 µg/ml), and CD43 (161-46, 1 µg/ml) for 6 h. Aggregation of cells in the absence of stimuli (normal conditions) was less than 4%. Percentage of aggregation was quantitatively determined by cell-cell adhesion assays. (B) Images of the aggregated cells in culture were obtained using an inverted phase-contrast microscope attached to a video camera, and captured using NIH image software. Results (aggregation relative to control culture in the presence of stimuli) are expressed as mean±SEM from three independent experiments performed in triplicate. **p<0.01 compared to the control group.

• 

  Fig. 3 Effect of blocking antibodies on cell-cell adhesion induced by ligation of surface adhesion molecules with aggregation-activating antibodies.(A) U937 cells were incubated with pro-aggregative (aggregation-activating) antibodies (1 µg/ml each as IgG1) to CD98 (AHN-18, 1 µg/ml), CD29 (MEM101A, 1 µg/ml), CD147 (M6-1D4, 10 µg/ml), and CD43 (161-46, 1 µg/ml) in the presence of aggregation-blocking antibodies to CD29 (P5D2, 1 µg/ml), CD98 (MEM 108, 1 µg/ml), and CD147 (MEM M6/1, 1 µg/ml) for 6 h. Aggregation of cells in the absence of stimuli (normal conditions) was less than 4%. Percentage of aggregation was quantitatively determined by cell-cell adhesion assays. (B) Images of the aggregated cells in culture were obtained using an inverted phase-contrast microscope attached to a video camera, and captured using NIH image software. Results (aggregation relative to control culture in the presence of stimuli) are expressed as mean±SEM from three independent experiments performed in triplicate. *p<0.05 and **p<0.01 compared to the control group.

• 

  Fig. 4 Effect of pharmacological inhibitors of ERK, PKCδ, and actin polymerization on cell-cell aggregation or cell-fibronectin adhesion induced by ligation of surface adhesion molecules with aggregation-activating antibodies or immobilized fibronectin.(A left panel) U937 cells were incubated with pro-aggregative (activating) antibodies (1 µg/ml each as IgG1) to CD98 (AHN-18, 1 µg/ml), CD29 (MEM101A, 1 µg/ml), CD147 (M6-1D4, 10 µg/ml), and CD43 (161-46, 1 µg/ml) in the presence of chemical inhibitors to ERK (U0126, 25 µM), PKCδ (rottlerin, 10 µM), and actin polymerization (Cyto B: cytochalasin B, 10 µM) for 3 h. Aggregation of cells in the absence of stimuli (normal conditions) was less than 4%. Percentage of aggregation was quantitatively determined by cell-cell adhesion assays. (A right panel) Images of the aggregated cells in culture were obtained using an inverted phase-contrast microscope attached to a video camera, and captured using NIH image software. (B) U937 cells pretreated with 10 µg/ml of function blocking antibody to CD29 (P5D2) or inhibitors [U0126 (20 µM) and cytochalasin B (10 µM)] were seeded on fibronectin (50 µg/ml)-immobilized plates and further incubated for 3 h. Attached cells were determined by crystal violet assay, as described in Materials and Methods. (C) Cytotoxic activity of U0126, rottlerin, and CytoB was confirmed by a conventional MTT assay. Results (aggregation relative to control culture in the presence of stimuli or % of cytotoxicity) are expressed as mean±SEM from three independent experiments performed in triplicate. **p<0.01 compared to the control group.

• 

  Fig. 5 Molecular association between CD29, CD98, CD187, and the actin cytoskeleton in U937 cell-cell adhesion.(A and C) U937 cells were incubated with pro-aggregative (activating) antibodies (1 µg/ml each as IgG1) to CD98 (AHN-18, 1 µg/ml), CD29 (MEM101A, 1 µg/ml), CD147 (M6-1D4, 10 µg/ml), and CD43 (161-46, 1 µg/ml) for 3 h. The localization patterns between CD29, CD98, CD147, and the actin cytoskeleton were evaluated by confocal microscopy. Results show one experiment out of three. (B) U937 cells (1×107 cells/ml) were treated with antibodies to CD147 (M6-1D4) and CD98 (AHN-18) for 6 h. After immunoprecipitation with antibodies to CD147 or CD98, the level of co-immunoprecipitated CD147 was determined by immunoblotting analysis with anti-CD147 antibodies. Results represent one experiment out of three. Relative intensities were calculated with the DNR Bioimaging system (Gelquant software Ver. 2.7).

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