Abstract
Endothelin 1 (ET-1) is a key regulator of vascular homeostasis. We have recently reported that the presence of Human antigen class I, HLA-B35, contributes to human dermal microvascular endothelial cell (HDMEC) dysfunction by upregulating ET-1 and proinflammatory genes. Likewise, a Toll-like receptor 3 (TLR3) ligand, Poly(I:C), was shown to induce ET-1 expression in HDMECs. The goal of this study was to determine the molecular mechanism of ET-1 induction by these two agonists. Because HLA-B35 expression correlated with induction of Binding Immunoglobulin Protein (BiP/GRP78) and several heat shock proteins, we first focused on ER stress and unfolded protein response (UPR) as possible mediators of this response. ER stress inducer, Thapsigargin (TG), HLA-B35, and Poly(I:C) induced ET-1 expression with similar potency in HDMECs. TG and HLA-B35 activated the PERK/eIF2α/ATF4 branch of the UPR and modestly increased the spliced variant of XBP1, but did not affect the ATF6 pathway. Poly(I:C) also activated eIF2α/ATF4 in a protein kinase R (PKR)-dependent manner. Depletion of ATF4 decreased basal expression levels of ET-1 mRNA and protein, and completely prevented upregulation of ET-1 by all three agonists. Additional experiments have demonstrated that the JNK and NF-κB pathways are also required for ET-1 upregulation by these agonists. Formation of the ATF4/c-JUN complex, but not the ATF4/NF-κB complex was increased in the agonist treated cells. The functional role of c-JUN in responses to HLA-B35 and Poly(I:C) was further confirmed in ET-1 promoter assays. This study identified ATF4 as a novel activator of the ET-1 gene. The ER stress/UPR and TLR3 pathways converge on eIF2α/ATF4 during activation of endothelial cells.
Highlights
Endothelin-1 (ET-1) is a potent vasoconstrictor and one of the key regulators of vascular homeostasis
In the initial experiment we compared the effects of HLA-B35 with a known Endoplasmic Reticulum (ER) stress inducer, thapsigargin (TG), and a Toll-like receptor 3 (TLR3) agonist, Poly(I:C), on the expression of ET-1 mRNA and protein in primary dermal microvascular endothelial cells (HDMECs)
In this study we show for the first time that ATF4 is a novel regulator of the ET-1 gene in endothelial cells
Summary
Endothelin-1 (ET-1) is a potent vasoconstrictor and one of the key regulators of vascular homeostasis. A complex network consisting of the common and tissue specific transcription factors responding in the coordinated fashion to physiological and pathological stimuli have been shown to regulate ET-1 expression in a cell type and context specific manner. One of the main regulatory factors is a FOS/JUN complex that binds to an activator protein 1 (AP-1) response element located at a -108 bp in the ET-1 promoter region. This site mediates upregulation of the ET-1 gene by phorbol esters, Angiotensin II, Thrombin, and High-density lipoprotein (HDL), which stimulate AP-1 in a Protein kinase C (PKC)-dependent manner. Other cell type specific response elements have been characterized [7]
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