Alpha2,6-sialic acid on platelet endothelial cell adhesion molecule (PECAM) regulates its homophilic interactions and downstream antiapoptotic signaling

Abstract

Antiangiogenesis therapies are now part of the standard repertoire of cancer therapies, but the mechanisms for the proliferation and survival of endothelial cells are not fully understood. Although endothelial cells are covered with a glycocalyx, little is known about how endothelial glycosylation regulates endothelial functions. Here, we show that alpha2,6-sialic acid is necessary for the cell-surface residency of platelet endothelial cell adhesion molecule (PECAM), a member of the immunoglobulin superfamily that plays multiple roles in cell adhesion, mechanical stress sensing, antiapoptosis, and angiogenesis. As a possible underlying mechanism, we found that the homophilic interactions of PECAM in endothelial cells were dependent on alpha2,6-sialic acid. We also found that the absence of alpha2,6-sialic acid down-regulated the tyrosine phosphorylation of PECAM and recruitment of Src homology 2 domain-containing protein-tyrosine phosphatase 2 and rendered the cells more prone to mitochondrion-dependent apoptosis, as evaluated using PECAM- deficient endothelial cells. The present findings open up a new possibility that modulation of glycosylation could be one of the promising strategies for regulating angiogenesis.

Figures

FIGURE 1.

Altered PECAM expression in ST6Gal I-deficient mice. A, brain sections from wild-type mice were stained with the Siaα2,6Gal-binding TJA-I lectin and an anti-PECAM antibody (MEC13.3) and then observed by confocal fluorescence microscopy. The colocalized staining appears yellow. B, brain sections from wild-type (WT) and ST6Gal I-deficient (KO) mice were stained with the TJA-I lectin, an anti-ST6Gal I antibody, and an anti-PECAM antibody (MEC13.3). Scale bars, 200 μm. C, lung homogenates prepared from wild-type and ST6Gal I-deficient mice were precipitated with SSA-agarose. The precipitated samples were analyzed by Western blotting with an anti-PECAM antibody (M-20). D, lung PECAM and GAPDH mRNA levels in wild-type and ST6Gal I-deficient mice were analyzed by real time PCR. The expression levels of PECAM were measured in duplicate and normalized by the corresponding GAPDH expression levels. Data are presented as means ± S.E. (n = 3). E, lung homogenates (10 μg of total protein) from wild-type and ST6Gal I-deficient mice were analyzed by immunoblotting with anti-PECAM (M-20) and anti-GAPDH antibodies. The bars represent the relative PECAM immunoreactivities shown as means ± S.E. (n = 6). *, p < 0.001.

FIGURE 2.

Increased cell-surface expression of PECAM after ST6Gal I expression. A, transfection of HUVECs was achieved by infection with adenovirus preparations of ST6Gal I (ST) or LacZ using 50 plaque-forming units/cell. At 48 h after infection, the cells (5 × 105) were detached, resuspended as single-cell suspensions, and probed with SSA-fluorescein isothiocyanate (FITC) or an anti-PECAM-PE antibody. B, lysates of HUVECs infected with ST6Gal I adenovirus (ST) or control adenovirus (LacZ) were analyzed by Western blotting using anti-PECAM and anti-GAPDH antibodies. The bars represent the relative immunoreactivities of PECAM to GAPDH shown as means ± S.E. (n = 3). C, cell-surface expressions of α2,6-sialic acid (detected by SSA-biotin and Alexa Fluor 488-conjugated streptavidin), PECAM, and VE-cadherin are shown as the mean fluorescence intensities (MFI) in HUVECs infected with ST6Gal I adenovirus or control adenovirus.

FIGURE 3.

Altered PECAM expression in ST6Gal I-deficient endothelial cells. A, primary liver sinusoidal endothelial cells were prepared from wild-type (WT) and ST6Gal I-deficient (KO) mice, fixed, and stained with an anti-PECAM antibody (green) and antibodies against a trans-Golgi marker (γ-adaptin; red) or an early endosome marker (EEA-1; red). Scale bar, 20 μm. B, primary liver sinusoidal endothelial cells from ST6Gal I-deficient mice were infected with adenovirus preparations for ST6Gal I (ST-Ad) or LacZ (LacZ-Ad) using 50 plaque-forming units/cell. 48 h after infection, the cells were fixed and stained with SSA lectin (green), an anti-PECAM antibody (MEC13.3, red), and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 20 μm. C, half-lives of PECAM cell-surface expression on endothelial cells from wild-type and ST6Gal I-deficient mice were determined after cell-surface biotinylation and incubation for 3, 6, 12, and 24 h. Data are presented as the means ± S.E. from three separate experiments.

FIGURE 4.

ST6Gal I expression is necessary for PECAM to transduce inhibitory signals. A and B, PECAM complexes were precipitated from lung lysates from wild-type (WT) and ST6Gal I-deficient (KO) mice using an anti-PECAM antibody and analyzed with anti-PECAM and anti-phosphotyrosine (pY) antibodies (A) or anti-SHP2 antibodies (B). C, endothelial cell lysates (20 μg of protein) from wild-type and ST6Gal I-deficient mice were analyzed by Western blotting using an anti-phosphotyrosine (pY) antibody. D, primary liver sinusoidal endothelial cells from the livers of wild-type and ST6Gal I-deficient mice were grown on 96-well plates. At 0, 2, 4, and 8 h after the addition of staurosporine (STSP), the cells were measured for their caspase-3/-7 activities using a multiwell luminometer. Data are presented as the means ± S.E. from three separate experiments. **, p < 0.0005, versus ST6Gal I-deficient mice. IP, immunoprecipitated.

FIGURE 5.

Sialic acid-based homophilic PECAM interactions. A, purities of PECAM-Fc and PECAM-His were verified by silver staining. B, PECAM-Fc absorbed to protein A-Sepharose was incubated with His-tagged PECAM in the presence or absence of V. cholerae sialidase. Alternatively, sialidase-treated PECAM-Fc was incubated with PECAM-His. The absorbed proteins on the protein A-Sepharose were analyzed by Western blotting with anti-human IgG antibody (for PECAM-Fc) or anti-His antibody (for PECAM-His). C, after PECAM-Fc (WT) or PECAMR90A-Fc (R90A) was incubated with PECAM-His, the absorbed proteins on the protein A-Sepharose were analyzed by Western blotting. D, HUVECs were stained with SSA lectin and an anti-PECAM antibody (H-200). Scale bar, 20 μm. E, fluorescence intensity profiles were taken along the line shown in the image of HUVECs stained with SSA lectin and an anti-PECAM antibody. The bars represent the signal intensities of the PECAM and SSA epitopes at junctional (j) and peripheral (p) areas shown as means ± S.E. (n = 6). *, p < 0.005; **, p < 0.05. DAPI, 4,6-diamidino-2-phenylindole.

FIGURE 6.

Schematic diagrams of a model for sialic acid-dependent homophilic PECAM interactions. PECAM proteins modified with sialic acids interact with one another at the cell surface, and subsequently recruit SHP2 to transduce inhibitory signals to the cell (left). Without the sialic acids, PECAM fails to mediate the homophilic interactions required for the underlying signal transduction (right). TM, transmembrane.