ATF4 is a novel regulator of MCP-1 in microvascular endothelial cells.
BACKGROUND
Monocyte chemoattractant protein-1 (MCP-1) is a major chemokine that recruits monocyte/macrophage to the site of tissue injury and plays a critical role in microvascular complications of diabetes. However, the mechanisms underlying the regulation of MCP-1 are not fully understood. The present study aims to explore the role of activating transcription factor 4 (ATF4), an ER stress-inducible transcription factor, in regulation of MCP-1 expression and production in brain and retinal microvascular endothelial cells.


METHODS
For in vitro study, primary brain microvascular endothelial cells isolated from ATF4 knockout mice or mouse retinal endothelial cells were treated with lipopolysaccharide (LPS) to induce MCP-1 expression. ATF4 expression/function was manipulated by adenoviruses expressing wild type ATF4 (Ad-ATF4) or a dominant negative mutant of the protein (Ad-ATF4DN). For in vivo study, MCP-1 expression was induced by intravitreal injection of LPS or Ad-ATF4 in heterozygous ATF4 knockout or wild type mice.


RESULTS
LPS treatment induced a dose- and time-dependent increase in ATF4 expression, ER stress and MCP-1 production in brain and retinal microvascular endothelial cells. Overexpression of ATF4 in endothelial cells significantly increased the secretion of MCP-1 and promoted THP-1 monocyte-endothelial cell adhesion. Conditioned medium from ATF4-overexpressiing endothelial cells significantly enhanced THP-1 cell migration. Consistently, intravitreal injection of Ad-ATF4 remarkably enhanced retinal levels of MCP-1 and promoted inflammatory cell infiltration into the vitreous and retina. In contrast, LPS-induced MCP-1 upregulation was markedly attenuated in ATF4-deficient endothelial cells and in retinas of ATF4 knockout mice, suggesting that ATF4 is essential for LPS-induced MCP-1 production in endothelial cells and in the retina. Mechanistically, overexpression of ATF4 enhanced, while inhibition of ATF4, attenuated the basal and LPS-stimulated phosphorylation of NF-κB, P38, and JNK. Furthermore, pharmacological inhibition of NF-κB, P38, or JNK significantly reduced ATF4-stimulated MCP-1 secretion from endothelial cells.


CONCLUSIONS
Taken together, our results suggest a critical role of ATF4 in the regulation of MCP-1 production in retinal and brain microvascular endothelial cells, which may contribute to inflammation-related endothelial injury in diseases such as diabetic retinopathy.
Departments of Ophthalmology and Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, The State University of New York, Buffalo, NY 14214 USA ; SUNY Eye Institute, The State University of New York, Buffalo, NY 14214 USA ; Department of Endocrinology, The 2nd Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian China.
Huibin
H
Huang
Department of Medicine, Endocrinology and Diabetes, Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma, 73104 OK USA.
Guangjun
G
Jing
Departments of Ophthalmology and Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, The State University of New York, Buffalo, NY 14214 USA ; SUNY Eye Institute, The State University of New York, Buffalo, NY 14214 USA ; Department of Medicine, Endocrinology and Diabetes, Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma, 73104 OK USA.
Joshua J
JJ
Wang
Department of Ophthalmology and Visual Sciences, University of Wisconsin, School of Medicine and Public Health, Madison, Wisconsin USA ; McPherson Eye Research Institute, University of Wisconsin, School of Medicine and Public Health, Madison, WI 53705 USA.
Nader
N
Sheibani
Departments of Ophthalmology and Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, The State University of New York, Buffalo, NY 14214 USA ; SUNY Eye Institute, The State University of New York, Buffalo, NY 14214 USA ; Department of Medicine, Endocrinology and Diabetes, Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma, 73104 OK USA.
Sarah X
SX
Zhang
Activating transcription factor 4; Microvascular endothelial cells; Monocyte chemoattractant protein 1