Sphingosine-1-phosphate receptor-2 mediated NFκB activation contributes to tumor necrosis factor-α induced VCAM-1 and ICAM-1 expression in endothelial cells

doi:10.1016/j.prostaglandins.2013.06.001

Prostaglandins & Other Lipid Mediators

Volume 106, October 2013, Pages 62-71

https://doi.org/10.1016/j.prostaglandins.2013.06.001 Get rights and content

Highlights

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TNFα treatment induces S1P₂ expression in endothelial cells.
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Ectopic expression of S1P₂ induces VCAM-1/ICAM-1 expression following S1P treatment in endothelial cells.
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S1P/S1P₂ signaling activates NFκB.
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NFκB inhibition abrogates S1P/S1P₂-induced ICAM-1/VCAM-1 expression.
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Inhibition of S1P₂ signaling diminishes TNFα-induced NFκB activation and VCAM-1/ICAM-1 expression in endothelial cells.

Abstract

Sphingosine-1-phosphate (S1P) regulates a wide array of biological functions in endothelial cells. We previously showed that S1P receptor subtype 2 (S1P₂) is significantly up-regulated in the atherosclerotic endothelium (J. Biol. Chem. 283:30363, 2008). In this study, we investigated the roles of S1P₂-mediated signaling in the proinflammatory responses of endothelial cells. Treatment with tumor necrosis factor-α (TNFα), a proinflammatory cytokine, increased the expression of S1P₂ receptors in endothelial cells. TNFα treatment also enhanced sphingosine kinase 1 expression and increased S1P production. Pharmacological inhibition or knockdown of S1P₂ receptors completely abrogated the TNFα-induced VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) expression in endothelial cells. In contrast, pharmacological inhibition or knockdown of other S1P receptor subtypes had no effect on the TNFα-stimulated ICAM-1 and VCAM-1 expression. Moreover, ectopic expression of S1P₂ receptors increased VCAM-1 and ICAM-1 expression in endothelial cells in response to S1P stimulation. Mechanistically, we show that antagonizing S1P₂ signaling markedly inhibited the TNFα-stimulated NFκB activation. Utilizing the NFκB reporter luciferase assay, the S1P/S1P₂ signaling was shown to stimulate NFκB activation. Moreover, the S1P/S1P₂-stimulated VCAM-1/ICAM-1 expression was completely abolished by the pharmacological inhibitor of NFκB. Collectively, our data suggest that TNFα treatment activates autocrine S1P/S1P₂ signaling, which subsequently activates NFκB and leads to the proinflammatory responses in endothelial cells.

Introduction

Atherosclerosis, a chronic inflammatory process, is characterized by the thickening of the arterial wall and is the primary cause of cardiovascular disease and cerebrovascular accident, two of the most common causes of illness and death worldwide [1]. An important risk factor for developing atherosclerosis is vascular inflammation, which involves the complex interactions of modified lipoproteins, monocyte-derived macrophages or foam cells, T lymphocytes, endothelial cells, and smooth muscle cells [2], [3].

Spingosine-1-phosphate (S1P), a serum-borne bioactive lipid mediator synthesized by sphingosine kinase (SphK), regulates an array of biological activities in various cell types [4], [5], [6], [7]. S1P can function either as an extracellular ligand or intracellular mediator [8], [9], [10]. When functioning as an extracellular ligand, S1P-regulated biological activities are mediated by the S1P family of G protein-coupled receptors (GPCRs) [8], [11], [12]. There are five identified members of the S1P receptor family: S1P₁, S1P₂, S1P₃, S1P₄, and S1P₅ (previous nomenclature: EDG-1, -5, -3, -6, -8, respectively) [13]. S1P receptor subtypes couple to different G_α polypeptides to regulate distinct signaling pathways [14], [15], [16]. Various S1P receptors are expressed in distinct combinations in different cell types to produce the appropriate biological effects. For example, S1P₁, S1P₂, and S1P₃ receptors are expressed in endothelial cells (ECs) [17], [18]. The signaling pathways regulated by S1P₁ and S1P₃ are required for cellular chemotaxis, adherens junction assembly, endothelial morphogenesis, and angiogenic response [17], [19]. Moreover, the balance of S1P₁ and S1P₂ signaling contributes to vascular integrity in vivo [20]. Disturbance of this balance, i.e., the up-regulation of S1P₂ signaling, may have functional implications in vascular dysfunctions such as endothelial senescence and atherosclerosis [18].

It has been shown that sphingosine kinase (SphK) plays a role in inflammatory disorders, such as rheumatoid arthritis [21], [22]. However, the molecular details of S1P signaling in the regulation of inflammation remain to be elucidated. Recent studies demonstrated that S1P₂ deficiency led to inhibition of macrophage proinflammatory responses and atherosclerosis in apoE-deficient mice [23]. Moreover, it was shown that S1P₃ mediates the chemotactic effect of S1P in macrophages in vitro and in vivo, and plays a causal role in atherosclerosis by causing inflammatory monocyte/macrophage recruitment and altering smooth muscle cell behavior [24]. These studies suggest that S1P signaling cascades play an important role in the development of atherosclerosis.

However, how S1P signaling contributes to atherosclerosis and vascular inflammation, particularly in the context of the endothelium, is poorly understood. We previously showed that senescence-associated endothelial dysfunctions can be attributed to the up-regulation of S1P₂ signaling [18]. We also found that S1P₂ receptors were markedly increased in atherosclerotic endothelium [18]. These results suggest that the up-regulated endothelial S1P₂ signaling may play a role in mediating endothelial proinflammatory responses and atherosclerosis development. Therefore, we examined roles of S1P signaling in ECs for proinflammatory cytokine-induced endothelial inflammation in this study. We observed that TNFα treatment increased the expression of S1P₂ receptors and production of S1P in ECs. More importantly, inhibition of endothelial S1P₂ signaling significantly diminished the TNFα-induced activation of NFκB and expression of proinflammation-associated adhesion molecules (i.e. VCAM-1, ICAM-1) in ECs. Furthermore, S1P₂ signaling is capable of activating NFκB directly. Also, S1P stimulation increases the expression of VCAM-1/ICAM-1 in ECs, ectopically expressing S1P₂ receptors. Collectively, our results suggest that S1P₂ signaling plays an important role in proinflammatory cytokine induced endothelial inflammation.

Section snippets

Reagents

Sphingosine-1-phosphate was purchased from Biomol; JTE-013 and VPC23019 were from Avanti. BAY 11-7085 was from Calbiochem EMD. Mouse-derived S1P₁ monoclonal antibody (E1-49) was generated by conventional hybridoma technology using bacteria expressed S1P₁ as an immunogen [25], [26]. E1-49 was shown to specifically react with the human S1P₁ receptor by immunostaining, immunoblotting, and immunoprecipitation [20], [25], [26]. Polyclonal anti-S1P₂ and anti-S1P₃ were from Cayman Chemical. Monoclonal

TNFα treatment enhances the expression of S1P₂, VCAM-1, and ICAM-1, and activates SphK1 in endothelial cells

Our previous study showed that S1P₂ was markedly increased in the lesion region of the atherosclerotic endothelium [18]. To investigate the role of S1P receptor subtypes in endothelial inflammation, cultured endothelial cells were treated with proinflammatory cytokine TNFα for 4 h. The expression of S1P receptor subtypes was assessed by real-time PCR. As shown in Fig. 1A, S1P₂ expression was significantly elevated by 2.3-fold at messenger RNA level (p < 0.01, t-test). The expression of S1P₁ and S1P

Discussion

Endothelial inflammation is an important risk factor in the initiation and development of atherosclerosis [2], [3]. Therefore, the specific signaling cascades that regulate endothelial inflammation, and the patho-physiological outcomes mediated by these endothelial proinflammatory signals are important questions in vascular biology. TNFα is a critical proinflammatory cytokine, and its inhibition is currently being used to treat various inflammatory and autoimmune disorders such as rheumatoid

Conclusion

TNFα treatment increased the expression of S1P₂ receptors and production of S1P in ECs. Inhibition of endothelial S1P₂ signaling significantly diminished the TNFα-induced activation of NFκB and expression of proinflammation-associated adhesion molecules in ECs. Our results highlight a novel role of S1P₂ signaling in the proinflammatory responses of the endothelium. Moreover, it was demonstrated that S1P₂ signaling plays an important role in the retention and pro-inflammation of macrophages

Acknowledgements

This work is supported by NIH grant R01HL071071 (M.L.) and by a Pilot and Feasibility Grant from the Michigan Diabetes Research and Training Center (NIH Grant 5P600-DK020572).

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Original research articleSphingosine-1-phosphate receptor-2 mediated NFκB activation contributes to tumor necrosis factor-α induced VCAM-1 and ICAM-1 expression in endothelial cells

Highlights

Abstract

Introduction

Section snippets

Reagents

TNFα treatment enhances the expression of S1P2, VCAM-1, and ICAM-1, and activates SphK1 in endothelial cells

Discussion

Conclusion

Acknowledgements

Exp Cell Res

J Biol Chem

J Biol Chem

J Biol Chem

Cell

J Biol Chem

Mol Cell

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Biochem Biophys Res Commun

J Biol Chem

J Biol Chem

Curr Opin Immunol

J Biol Chem

FEBS Lett

J Biol Chem

J Biol Chem

Translating molecular discoveries into new therapies for atherosclerosis

Nature

Inflammation in atherosclerosis

Nature

Endothelial-dependent mechanisms of leukocyte recruitment to the vascular wall

Circ Res

Sphingosine 1-phosphate: a prototype of a new class of second messengers

J Leukoc Biol

Sphingosine 1-phosphate is a blood constituent released from activated platelets, possibly playing a variety of physiological and pathophysiological roles

Acta Biochim Pol

Sphingosine-1-phosphate: extracellular mediator or intracellular second messenger?

Biochem Pharmacol

Original research article
Sphingosine-1-phosphate receptor-2 mediated NFκB activation contributes to tumor necrosis factor-α induced VCAM-1 and ICAM-1 expression in endothelial cells

TNFα treatment enhances the expression of S1P₂, VCAM-1, and ICAM-1, and activates SphK1 in endothelial cells