Abstract
BackgroundDespite its first discovery by in silico cloning of novel endothelial cell-specific genes a decade ago, the biological functions of endothelial cell-specific molecule 2 (ECSM2) have only recently begun to be understood. Limited data suggest its involvement in cell migration and apoptosis. However, the underlying signaling mechanisms and novel functions of ECSM2 remain to be explored.Methodology/Principal FindingsA rabbit anti-ECSM2 monoclonal antibody (RabMAb) was generated and used to characterize the endogenous ECSM2 protein. Immunoblotting, immunoprecipitation, deglycosylation, immunostaining and confocal microscopy validated that endogenous ECSM2 is a plasma membrane glycoprotein preferentially expressed in vascular endothelial cells (ECs). Expression patterns of heterologously expressed and endogenous ECSM2 identified that ECSM2 was particularly concentrated at cell-cell contacts. Cell aggregation and transwell assays showed that ECSM2 promoted cell-cell adhesion and attenuated basic fibroblast growth factor (bFGF)-driven EC migration. Gain or loss of function assays by overexpression or knockdown of ECSM2 in ECs demonstrated that ECSM2 modulated bFGF-directed EC motility via the FGF receptor (FGFR)-extracellular regulated kinase (ERK)-focal adhesion kinase (FAK) pathway. The counterbalance between FAK tyrosine phosphorylation (activation) and ERK-dependent serine phosphorylation of FAK was critically involved. A model of how ECSM2 signals to impact bFGF/FGFR-driven EC migration was proposed.Conclusions/SignificanceECSM2 is likely a novel EC junctional protein. It can promote cell-cell adhesion and inhibit bFGF-mediated cell migration. Mechanistically, ECSM2 attenuates EC motility through the FGFR-ERK-FAK pathway. The findings suggest that ECSM2 could be a key player in coordinating receptor tyrosine kinase (RTK)-, integrin-, and EC junctional component-mediated signaling and may have important implications in disorders related to endothelial dysfunction and impaired EC junction signaling.
Highlights
Angiogenesis is essential for normal organ growth, development and wound healing, and an important determinant for many diseases such as cancer, atherosclerosis, diabetic retinopathies, and rheumatoid arthritis [1,2]
Bioinformatics analysis and heterologous expression of GFP, myc, or FLAG-tagged endothelial cell-specific molecule 2 (ECSM2) proteins in several mammalian cell systems further suggested that ECSM2 is a cell membrane protein consisting of an N-terminal extracellular domain (ECD), a single transmembrane domain (TM), and a small, highly conserved Cterminal intracellular domain (ICD) [3,4,6]
The cell membrane localization of endogenous ECSM2 in HUVEC can be detected by immunostaining with this rabbit anti-ECSM2 monoclonal antibody (RabMAb) and confocal microscopy (Figure 1F), which confirmed the previous findings of heterologously expressed tagged ECSM2 proteins [3,4,6]
Summary
Angiogenesis is essential for normal organ growth, development and wound healing, and an important determinant for many diseases such as cancer, atherosclerosis, diabetic retinopathies, and rheumatoid arthritis [1,2]. There has been a long-standing interest in identifying genes or preferentially expressed in ECs and understanding their biological functions. This may lead to the discovery of new pathways and molecular targets with therapeutic potentials. Endothelial cellspecific molecule 2 (ECSM2) [3,4], known as endothelial cellspecific chemotaxis receptor (ECSCR) [5] and apoptosis regulator through modulating cIAP expression (ARIA) [6], was initially identified a decade ago by in silico cloning of novel EC-specific genes [7]. Despite its first discovery by in silico cloning of novel endothelial cell-specific genes a decade ago, the biological functions of endothelial cell-specific molecule 2 (ECSM2) have only recently begun to be understood. The underlying signaling mechanisms and novel functions of ECSM2 remain to be explored
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