CD93 promotes β1 integrin activation and fibronectin fibrillogenesis during tumor angiogenesis
Roberta Lugano, Kalyani Vemuri, Di Yu, Michael Bergqvist, Anja Smits, Magnus Essand, Staffan Johansson, Elisabetta Dejana, Anna Dimberg
Roberta Lugano, Kalyani Vemuri, Di Yu, Michael Bergqvist, Anja Smits, Magnus Essand, Staffan Johansson, Elisabetta Dejana, Anna Dimberg
View: Text | PDF
Research Article Oncology Vascular biology

CD93 promotes β1 integrin activation and fibronectin fibrillogenesis during tumor angiogenesis

Abstract

Tumor angiogenesis occurs through regulation of genes that orchestrate endothelial sprouting and vessel maturation, including deposition of a vessel-associated extracellular matrix. CD93 is a transmembrane receptor that is upregulated in tumor vessels in many cancers, including high-grade glioma. Here, we demonstrate that CD93 regulates β1 integrin signaling and organization of fibronectin fibrillogenesis during tumor vascularization. In endothelial cells and mouse retina, CD93 was found to be expressed in endothelial filopodia and to promote filopodia formation. The CD93 localization to endothelial filopodia was stabilized by interaction with multimerin-2 (MMRN2), which inhibited its proteolytic cleavage. The CD93-MMRN2 complex was required for activation of β1 integrin, phosphorylation of focal adhesion kinase (FAK), and fibronectin fibrillogenesis in endothelial cells. Consequently, tumor vessels in gliomas implanted orthotopically in CD93-deficient mice showed diminished activation of β1 integrin and lacked organization of fibronectin into fibrillar structures. These findings demonstrate a key role of CD93 in vascular maturation and organization of the extracellular matrix in tumors, identifying it as a potential target for therapy.

Authors

Roberta Lugano, Kalyani Vemuri, Di Yu, Michael Bergqvist, Anja Smits, Magnus Essand, Staffan Johansson, Elisabetta Dejana, Anna Dimberg

×

Figure 3

MMRN2 downregulation impairs CD93 protein stability in endothelial cells.

Options: View larger image (or click on image) Download as PowerPoint
MMRN2 downregulation impairs CD93 protein stability in endothelial cells...
(A) Western blot and real-time quantitative PCR (qPCR) showing the efficiency of CD93 knockdown in HDBECs (siCD93_1 and siCD93_2) at 48 hours after siRNA transfection. Untransfected cells (Mock) and scramble siRNA-transfected cells (siCtrl) were used as controls. (B) Western blot and real-time qPCR for MMRN2 showing the efficiency of MMRN2 knockdown in HDBECs (siMMRN2_3 and siMMRN2_6). (C) MMRN2 mRNA levels after CD93 knockdown in HDBECs. (D) CD93 mRNA level after MMRN2 knockdown. Values represent fold change relative to siCtrl-transfected cells. (E) Western blot against MMRN2 in siCD93-transfected cells and controls. β-Actin was used as loading control. (F) Western blot against CD93 and β-actin in siMMRN2-transfected cells and controls. (G) Western blot against CD93 in control and siMMRN2-silenced HDBECs in the presence or absence of 50 μg/ml cycloheximide (CHX). The stability of CD93 in control cells and in siMMRN2-silenced cells was assessed at different time points after CHX treatment (0, 30, 60, and 240 minutes). (H) Quantification of CD93 levels during specific time points in the presence of 50 μg/ml CHX in control cells and siMMRN2-silenced cells. Values represent the percentage of CD93 relative to CHX-untreated cells in all conditions (control and siMMRN2 cells). *P < 0.05; 2-way ANOVA with Tukey’s post-test. Values represent mean ± SEM (3 independent experiments). (I) Representative Western blot of soluble CD93 (sCD93) levels in conditioned media supplemented with 1%FBS (EBM 1%FBS) derived from control and siMMRN2-silenced cells. (J) Quantification of sCD93 levels in the conditioned media derived from control and siMMRN2-treated cells. Values shown in the quantification graphs in A–F and J represent mean ± SEM (3 independent experiments); *P < 0.05, **P < 0.001; 1-way ANOVA with Dunnett’s multiple-comparisons test.