CREG Promotes the Proliferation of Human Umbilical Vein Endothelial Cells through the ERK/Cyclin E Signaling Pathway

Jie Tao,Mingyu Sun,Xiaoxiang Tian,Xinliang Ma,Yang Li,Shaowei Liu,Yaling Han,Jian Zhang,Chenghui Yan

doi:10.3390/ijms140918437

Abstract

Cellular repressor of E1A-stimulated genes (CREG) is a recently discovered secreted glycoprotein involved in homeostatic modulation. We previously reported that CREG is abundantly expressed in the adult vascular endothelium and dramatically downregulated in atherosclerotic lesions. In addition, CREG participates in the regulation of apoptosis, inflammation and wound healing of vascular endothelial cells. In the present study, we attempted to investigate the effect of CREG on the proliferation of vascular endothelial cells and to decipher the underlying molecular mechanisms. Overexpression of CREG in human umbilical vein endothelial cells (HUVEC) was obtained by infection with adenovirus carrying CREG. HUVEC proliferation was investigated by flow cytometry and 5-bromo-2′-deoxy-uridine (BrdU) incorporation assays. The expressions of cyclins, cyclin-dependent kinases and signaling molecules were also examined. In CREG-overexpressing cells, we observed a marked increase in the proportion of the S and G2 population and a decrease in the G0/G1 phase population. The number of BrdU positively-stained cells also increased, obviously. Furthermore, silencing of CREG expression by specific short hairpin RNA effectively inhibited the proliferation of human umbilical vein endothelial cells (HUVEC). CREG overexpression induced the expression of cyclin E in both protein and mRNA levels to regulate cell cycle progression. Further investigation using inhibitor blocking analysis identified that ERK activation mediated the CREG modulation of the proliferation and cyclin E expression in HUVEC. In addition, blocking vascular endothelial growth factor (VEGF) in CREG-overexpressed HUVEC and supplementation of VEGF in CREG knocked-down HUVEC identified that the pro-proliferative effect of CREG was partially mediated by VEGF-induced ERK/cyclin E activation. These results suggest a novel role of CREG to promote HUVEC proliferation through the ERK/cyclin E signaling pathway.

Highlights

During vascular development and pathological angiogenesis, the maintenance of blood vessel homeostasis and its functional execution depend on the integrity of vascular endothelium [1], which is affected by proliferation, migration and apoptosis of endothelial cells
These results suggest a novel role of Cellular repressor of E1A-stimulated genes (CREG) to promote human umbilical vein endothelial cells (HUVEC) proliferation through the ERK/cyclin E signaling pathway
AdCREG viral transduction led to a ~6.2-fold increase in CREG expression in HUVEC-AdCREG compared with the HUVEC-AdGFP group (Figure 1B)

Summary

Introduction

During vascular development and pathological angiogenesis, the maintenance of blood vessel homeostasis and its functional execution depend on the integrity of vascular endothelium [1], which is affected by proliferation, migration and apoptosis of endothelial cells. Cellular repressor of E1A-stimulated genes (CREG), originally reported as an antagonist of transcriptional activation and cellular transformation by E1A in 1998 [3], was recently identified as a secreted glycoprotein [4,5] and a lysosomal protein [6] It is widely expressed in adult tissues, but much less so in undifferentiated cells [4]. Further studies showed that CREG can induce endothelial cell migration by activating the ILK/Akt/mTOR/vascular endothelial growth factor (VEGF) 165 signaling pathway [10] and attenuate atherosclerotic endothelium apoptosis via the VEGF/PI3K/Akt pathway [9]. These series of observations suggest that CREG may play an important role in adult neovascularization and endothelial homeostasis. We investigated the effect of CREG on vascular endothelial cell cycle dynamics and the possible molecular mediators

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Conclusion