COSMC Is Overexpressed in Proliferating Infantile Hemangioma and Enhances Endothelial Cell Growth via VEGFR2

<div><p>Infantile hemangiomas are localized lesions comprised primarily of aberrant endothelial cells. COSMC plays a crucial role in blood vessel formation and is characterized as a molecular chaperone of T-synthase which catalyzes the synthesis of T antigen (Galβ1,3GalNAc). T antigen expression is associated with tumor malignancy in many cancers. However, roles of COSMC in infantile hemangioma are still unclear. In this study, immunohistochemistry showed that COSMC was upregulated in proliferating hemangiomas compared with involuted hemangiomas. Higher levels of T antigen expression were also observed in the proliferating hemangioma. Overexpression of COSMC significantly enhanced cell growth and phosphorylation of AKT and ERK in human umbilical vein endothelial cells (HUVECs). Conversely, knockdown of COSMC with siRNA inhibited endothelial cell growth. Mechanistic investigation showed that O-glycans were present on VEGFR2 and these structures were modulated by COSMC. Furthermore, VEGFR2 degradation was delayed by COSMC overexpression and facilitated by COSMC knockdown. We also showed that COSMC was able to regulate VEGF-triggered phosphorylation of VEGFR2. Our results suggest that COSMC is a novel regulator for VEGFR2 signaling in endothelial cells and dysregulation of COSMC expression may contribute to the pathogenesis of hemangioma.</p> </div

COSMC is overexpressed in proliferating hemangiomas.

<p>(A) Immunohistochemistry of human hemangioma tissues. Paraffin-embedded hemangiomas in proliferating (N = 13), involuting (N = 21), and involuted (N = 9) phases were immunostained with anti-COSMC antibody or biotin-conjugated peanut agglutinin (PNA). Representative images are shown. Scale bars: 50 µm. Negative controls did not show specific staining (data not shown). (B) COSMC is overexpressed in proliferating and involuting hemangiomas compared with involuted hemangiomas. Intensity of immunostaining was quantified. N = patient numbers. Data are presented as means ± SEM. **<i>P</i><0.01; ***<i>P</i><0.001.</p

COSMC overexpression modulates O-glycans on VEGFR2.

<p>(A) Changes in O-glycans on VEGFR2 in COSMC overexpressing HUVECs. Cell lysates of HUVECs transfected with control or <i>COSMC</i> plasmids were treated with or without neuraminidase, pulled down with VVA or PNA lectins, then immunoblotted with anti-VEGFR2 antibody. β-actin is an internal control. (B) COSMC knockdown in HUVECs decreases binding of PNA to VEGFR2. (C) O-glycans are present on VEGFR2 in human primary hemangiomas. Tissue lysates of proliferating and involuted hemangiomas with (+) or without (−) neuraminidase treatment were pulled down (PD) by PNA and then immunoblotted with anti-VEGFR2 antibody. β-actin is an internal control. (D) COSMC overexpression enhances phosphorylation of VEGFR2 in HUVECs. HUVECs were serum starved for 4 h and then treated with 20 ng/ml of VEGF for different time periods. Phosphorylation of VEGFR2, AKT, and ERK were analyzed by Western blotting. β-actin is a loading control. Representative images from two independent experiments were shown. Signals on Western blots were quantified by ImageQuant5.1. (E) COSMC knockdown suppresses phosphorylation of VEGFR2 in HUVECs. Signals on Western blots were quantified by ImageQuant5.1.</p

Expression of COMSC in HUVECs and EA.hy926 cells.

<p>(A) Proliferating infantile hemangiomas (IHs) (n = 3 patients) express higher levels of COSMC than HUVECs (n = 3 batches). <i>COSMC</i> mRNA levels were analyzed by real-time RT-PCR. *<i>P</i><0.05. In lower panel, <i>COSMC</i> is overexpressed in HUVECs. (B) Western blots showing COSMC overexpression (left panel) and knockdown (right panel) in HUVECs. β-actin is a loading control. Relative intensity of signals on Western blots was quantified by ImageQuant5.1. (C) COSMC overexpression and knockdown in human endothelial cell line EA.hy926. Relative intensity of signals on Western blots was quantified by ImageQuant5.1. (D) COSMC overexpression and knockdown modulate cell surface carbohydrates on HUVECs. Left panel, COSMC overexpression enhances T synthase and T antigen expression in HUVECs. HUVECs were cell surface biotinylated, lysed, pulled down (PD) with PNA, and then blotted with streptavidin-HRP. The arrow indicated that a protein band with molecular mass of 220-kDa has increased PNA binding in COSMC-transfected HUVECs. Cell lysates were Western blotted for detecting T synthase expression and β-actin was used as a loading control. Right panel, COSMC knockdown increased glycoproteins pulled down by VVA, which recognizes Tn antigen.</p

COSMC overexpression enhances cell growth in HUVECs.

<p>(A) Cell growth of HUVECs transfected with pcDNA3.1 control plasmid (open bars) or COSMC/pcDNA3.1 (closed bars) analyzed by trypan blue exclusion assays. (B) Cell growth of HUVECs transfected with control siRNA (open bars) or COSMC siRNA (closed bars) analyzed by trypan blue exclusion assays. (C) Cell growth of EA.hy926 cells overexpressing COSMC. (D) Cell growth of EA.hy926 cells with COSMC knockdown. Results are presented as means ± SD from three independent experiments. *<i>P</i><0.05 and **<i>P</i><0.01, compared with mock.</p

COSMC modulates protein degradation of VEGFR2.

<p>(A) COSMC overexpression delays degradation of VEGFR2. HUVECs were treated with cycloheximide (10 µg/ml) to block protein synthesis and 20 ng/ml of VEGF to trigger internalization and degradation of VEGFR2 for indicated time points. Upper panel shows representative Western blots. Lower panel shows signals on Western blots quantified by ImageQuant5.1 for HUVECs transfected with pcDNA3.1 control plasmid (dashed line) and UVECs transfected with COSMC/pcDNA3.1 (solid line). (B) COSMC knockdown facilitates degradation of VEGFR2. VEGFR2 degradation in HUVECs transfected with control siRNA (dashed line) or COSMC siRNA (solid line) was shown. Representative data from two independent experiments are presented.</p

Roles of AKT and ERK signaling pathways in COSMC-enhanced cell proliferation.

<p>(A) COSMC overexpression enhances phosphorylation of AKT and ERK. Western blotting was performed to analyze protein expression. Representative images are presented. Relative intensity of signals was quantified by ImageQuant5.1 and shown. (B) COSMC knockdown inhibits phosphorylation of AKT and ERK. (C) Effects of AKT and ERK inhibitors on cell proliferation. HUVECs transfected with mock or COSMC plasmids were treated with DMSO control, 5 µM of LY294002, or 10 µM of PD98059. Cell viability was analyzed by MTT assays at different time points. Results are presented as means ± SD from three independent experiments. *<i>P</i><0.05; **<i>P</i><0.01.</p

C1GALT1 regulates integrin β1 activity and signaling.

<p>(<i>a</i>) The activity of integrin β1 was enhanced in C1GALT1 overexpressing cells. Mock and C1GALT1 transfectants were analyzed by flow cytometry with anti-integrin β1 antibody (left) or anti-activated integrin β1 (HUTS-21) antibody (right). Non-specific mouse IgG was used as a background fluorescence control (dash line). The mean fluorescence intensities (MFI) are shown at the bottom. Results are represented as means ± SD from three independent experiments. * <i>P</i><0.05. (<i>b</i>) Knockdown of C1GALT1 suppressed integrin β1 activity. Control (Ctr sh) and C1GALT1 knockdown (C1GALT1 sh6 and C1GALT1 sh8) cells were analyzed by flow cytometry with the indicated antibodies. The average of MFI is shown at the bottom. Results are represented as means ± SD from three independent experiments. * <i>P</i><0.05. (<i>c</i>) C1GALT1 regulated integrin β1-induced FAK activation. p-FAK (Tyr397) and total FAK in C1GALT1 overexpressing cells (left) and knockdown cells (right) were analyzed by Western blotting. Transfectants were seeded on BSA or collagen IV (Col-IV)-coated plates for 0.5 h. The integrin β1 blocking antibody (P4C10, 2 µg/ml) was incubated with cells for 10 min before seeding on collagen IV-coated plates. GAPDH was used as a loading control.</p

C1GALT1 induces HCC cell adhesion, migration and invasion through integrin β1.

<p>(<i>a</i>) The effects of the integrin β1-blocking antibody, P4C10, on C1GALT1 overexpressing cells. HCC cells were pre-treated with the indicated concentration of P4C10 or control IgG for 10 min. C1GALT1-induced cell-collagen IV adhesion (left), migration (middle), and invasion (right) were suppressed by the integrin β1-blocking antibody. Results are represented as means ± SD from three independent experiments. **<i>P</i><0.01. (<i>b</i>) The effects of the integrin β1-blocking antibody on C1GALT1 knockdown cells. P4C10 inhibited cell-collagen IV adhesion (left), migration (middle), and invasion (right) in cells transfected with control siRNA (Ctr si). Results are shown as means ± SD. * <i>P</i><0.05; **<i>P</i><0.01.</p

C1GALT1 modifies O-glycans on integrin β1.

<p>(<i>a</i>) Integrin β1 carried O-glycans in HCC cells. Lysates of HA22T cells (0.5 mg) were treated with neuraminidase (Neu) and/or PNGaseF and then pulled down (PD) by PNA agarose beads. The pulled down proteins were separated by 6% SDS-PAGE and analyzed by immunoblotting (IB) with anti-integrin β1 antibody. (<i>b</i>) C1GALT1 enhanced PNA binding to integrin β1 in HCC36 cells. (<i>c</i>) Knockdown of C1GALT1 suppressed PNA binding to integrin β1 in HA22T cells. Cell lysates (1.2 mg) were immunoprecipitated (IP) by anti-integrin β1 antibody, and were treated with (+) or without (−) neuraminidase. Proteins were separated by 8% SDS-PAGE, and then blotted with PNA or anti-integrin β1 antibody. Non-specific mouse IgG was used as control.</p