Plasma membrane microdomains regulate TACE‐dependent TNFR1 shedding in human endothelial cells

Alessio D.’Alessio,Antonio Filippini,Elio Ziparo,Bianca Esposito,Claudia Giampietri,Jordan S Pober

doi:10.1111/j.1582-4934.2011.01353.x

Abstract

Upon stimulation by histamine, human vascular endothelial cells (EC) shed a soluble form of tumour necrosis factor receptor 1 (sTNFR1) that binds up free TNF, dampening the inflammatory response. Shedding occurs through proteolytic cleavage of plasma membrane-expressed TNFR1 catalysed by TNF-α converting enzyme (TACE). Surface expressed TNFR1 on EC is largely sequestered into specific plasma membrane microdomains, the lipid rafts/caveolae. The purpose of this study was to determine the role of these domains in TACE-mediated TNFR1 shedding in response to histamine. Human umbilical vein endothelial cells derived EA.hy926 cells respond to histamine via H1 receptors to shed TNFR1. Both depletion of cholesterol by methyl-β-cyclodextrin and small interfering RNA knockdown of the scaffolding protein caveolin-1 (cav-1), treatments that disrupt caveolae, reduce histamine-induced shedding of membrane-bound TNFR1. Moreover, immunoblotting of discontinuous sucrose gradient fractions show that TACE, such as TNFR1, is present within low-density membrane fractions, concentrated within caveolae, in unstimulated EA.hy926 endothelial cells and co-immunoprecipitates with cav-1. Silencing of cav-1 reduces the levels of both TACE and TNFR1 protein and displaces TACE, from low-density membrane fractions where TNFR1 remains. In summary, we show that endothelial lipid rafts/caveolae co-localize TACE to surface expressed TNFR1, promoting efficient shedding of sTNFR1 in response to histamine.

Highlights

Binding of TNF to a variety of cell types activates signaling pathways that regulate several different processes of medical significance such as apoptosis, inflammation, immunity and metabolism and has been implicated in the pathogenesis of several diseases [1,2]
FACS analysis confirmed that both histamine and TMPH stimulation reduced the expression of TNFR1 on the plasma membrane (Fig. 1B), and that this effect was prevented by mepyramine (Fig. 1C), further supporting the involvement of only H1R in this mechanism
We conclude that EA.hy926 cells, like Human Umbilical Vein Endothelial Cells (HUVEC), respond to histamine through H1R to induce TACE-mediated shedding of TNFR1 from the plasma membrane generating a soluble form of the receptor

Summary

Introduction

Binding of TNF to a variety of cell types activates signaling pathways that regulate several different processes of medical significance such as apoptosis, inflammation, immunity and metabolism and has been implicated in the pathogenesis of several diseases [1,2]. The shedding of membrane TNFR1 (mTNFR1) act by reducing the amount of mTNFR1 and produces a soluble 27-30 kD form of the receptor (sTNFR1) which compete with mTNFR1 to reduce cell sensitivity to TNF [5,6]. Both in human umbilical vein (HUV)EC and in the EAhy.926 endothelial cell line, the Golgi apparatus plays an important role in this scenario, functioning as a intracellular reservoir of TNFR1 from which it can be translocated to the plasma membrane and shed in response to stimuli [6,7,8] such as histamine. In the present study we extended these findings and propose a key function of cav-1 in both the retaining of TACE within the caveolar network and the shedding of TNFR1 given that the disruption of caveolae inhibits histamine and H1Rmediated sTNFR1 release

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Conclusion