Galectin-3 interacts with the cell-surface glycoprotein CD146 (MCAM, MUC18) and induces secretion of metastasis-promoting cytokines from vascular endothelial cells

Abstract

The galactoside-binding protein galectin-3 is increasingly recognized as an important player in cancer development, progression, and metastasis via its interactions with various galactoside-terminated glycans. We have shown previously that circulating galectin-3, which is increased up to 30-fold in cancer patients, promotes blood-borne metastasis in an animal cancer model. This effect is partly attributable to the interaction of galectin-3 with unknown receptor(s) on vascular endothelial cells and causes endothelial secretion of several metastasis-promoting cytokines. Here we sought to identify the galectin-3-binding molecule(s) on the endothelial cell surface responsible for the galectin-3-mediated cytokine secretion. Using two different galectin-3 affinity purification processes, we extracted four cell membrane glycoproteins, CD146/melanoma cell adhesion molecule (MCAM)/MUC18, CD31/platelet endothelial cell adhesion molecule-1 (PECAM-1), CD144/VE-cadherin, and CD106/Endoglin, from vascular endothelial cells. CD146 was the major galectin-3-binding ligand and strongly co-localized with galectin-3 on endothelial cell surfaces treated with exogenous galectin-3. Moreover, galectin-3 bound to N-linked glycans on CD146 and induced CD146 dimerization and subsequent activation of AKT signaling. siRNA-mediated suppression of CD146 expression completely abolished the galectin-3-induced secretion of IL-6 and G-CSF cytokines from the endothelial cells. Thus, CD146/MCAM is the functional galectin-3-binding ligand on endothelial cell surfaces responsible for galectin-3-induced secretion of metastasis-promoting cytokines. We conclude that CD146/MCAM interactions with circulating galectin-3 may have an important influence on cancer progression and metastasis.

Keywords: CD146; IL-6; MCAM; carbohydrate-binding protein; cytokine; endothelium; galectin; galectin-3.

Figure 1.

Galectin-3 induces cytokine secretion from both human micro- and macrovascular endothelial cells. A and B, HMVEC-Ls (A) and HUVECs (B) were treated with BSA or galectin-3 for 24 h, and the concentrations of G-CSF, GM-CSF, IL-6, and ICAM-1 in the culture medium were determined. Data are presented as mean ± S.E. of at least three independent experiments, each in triplicate. ***, p < 0.001.

Figure 2.

Identification of endothelial cell surface galectin-3-binding proteins. A, galectin-3 blotting/overlay shows similar binding of galectin-3 to a number of proteins in HUVECs and HUVECs. MW, molecular weight. B, galectin-3 affinity purification and protein identification. HUVEC cell lysates were separated by SDS-PAGE and stained by silver staining (left panel) or applied to nickel- or agarose-conjugated galectin-3 columns. Bound proteins were released by lactose and identified by mass spectrometry or separated by SDS-PAGE and stained by silver staining (right panel).

Figure 3.

Identification of CD146/MCAM as the major galectin-3-binding protein. A and B, HUVECs were treated with galectin-3 (4 μg) for 60 min. The cells were then treated without (A) or with (B) DTSSP cross-linker before immunoprecipitation (IP) with antibodies against MCAM, PECAM-1, PE-Cadherin, or endoglin, followed by immunoblotting (IB) with anti-galectin-3 antibody and peroxidase-conjugated secondary antibody. Co-immunoprecipitated galectin-3 and the heavy (IgG-H) and light (IgG-L) chains of the antibody/immunoglobulin used in the immunoprecipitation are indicated by arrows. Additional arrows show nonspecific (NS) antibody/immunoglobulin binding. MW, molecular weight.

Figure 4.

Co-localization of exogenous galectin-3 with CD146/MCAM in HUVECs. After treatment of HUVECs without (top panel) or with (bottom panel) galectin-3 (4 μg/ml) for 60 min, the cells were fixed, and CD146/MCAM (green) and galectin-3 (red) immunohistochemistry was conducted and followed by confocal microscopy. Cell nuclei were stained by DAPI (blue).

Figure 5.

Galectin-3 binds to N-linked glycans on CD146/MCAM. After immunoprecipitation (IP) of HUVECs with anti-CD146 antibody, the immunoprecipitates were treated with PNGaseF or O-glycanase before electrophoresis and immunoblotting (IB) with CD146/MCAM and galectin-3 antibody. MW, molecular weight.

Figure 6.

Suppression of CD146/MCAM expression abolishes galectin-3-induced cytokine secretion. A–C, HUVECs were treated with control or CD146/MCAM siRNA for 36 h. The cells were then either lysed and analyzed for CD146/MCAM expression by CD146/MCAM immunoblotting (A) or further treated with galectin-3 (4 μg/ml) for 24 h before the levels of G-CSF (B) or IL-6 (C) in the culture medium were determined. *, p < 0.5 (unpaired t test).

Figure 7.

Galectin-3 induces CD146/MCAM dimerization and downstream AKT signaling. A–C, HUVECs were treated with galectin-3 (4 μg/ml) for various times. The cells were lysed and applied to SDS-PAGE under non-denatured (A) or denatured (B) conditions and probed by anti-CD146/MCAM antibody. Cellular proteins were also probed with antibodies against p-AKT and AKT (C). MW, molecular weight.

Figure 8.

Activation of multiple downstream signaling pathways. HUVECs were treated with BSA (top panel) or galectin-3 (4 μg/ml) (bottom panel) for 24 h before the cells were lysed and analyzed by the Profiler human phospho-kinase array.