The metalloprotease ADAM8 is associated with and regulates the function of the adhesion receptor PSGL‐1 through ERM proteins

Abstract

The P-selectin glycoprotein ligand-1 (PSGL-1) is involved in the initial contact of leukocytes with activated endothelium, and its adhesive function is regulated through its proteolytic processing. We have found that the metalloprotease ADAM8 is both associated with PSGL-1 through the ezrin-radixin-moesin actin-binding proteins and able to cause the proteolytic cleavage of this adhesion receptor. Accordingly, ADAM8 knockdown increases PSGL-1 expression, and functional assays show that ADAM8 is able to reduce leukocyte rolling on P-selectin and hence on activated endothelial cells. We conclude that ADAM8 modulates the expression and function of PSGL-1.Key words: ADAM8 . Adhesion . Cell migration . ERM . Neutrophil . PSGL-1 See accompanying Commentary by Zarbock and RossaintIntroduction ADAM family is a group of transmembrane glycoproteins that possess proteolytic and signaling properties and are implicated in both cell adhesion and cell fusion processes [1]. ADAMs are usually activated by furin or other convertases as well as autocatalytically, in the case of ADAM8 [2]. It has been described that ADAM8 cleaves important cell surface proteins [3,4], cytokines and growth factors [5]. ADAM8 is overexpressed under several pathological conditions involving inflammation and remodeling of the extracellular matrix, including malignant diseases and asthma [6][7][8].P-selectin glycoprotein ligand-1 (PSGL-1), through its interaction with P-, E-and L-selectins, mediates the tethering and rolling of leukocytes on endothelial cells prior to their extravasation [9,10], triggers the activation of transcription factors like cFos [11] in leukocytes and induces the generation of tolerogenic DCs which promote the differentiation of Treg cells [12]. Although it was described that PSGL-1 was a substrate of the proteases BACE1 and ADAM10 [13], neither the physiological context of cleavage nor the mechanism responsible for its shedding have been identified so far. In this work, we demonstrate that in leukocytes SHORT COMMUNICATION Ã These authors contributed equally to this work. 3436Frontline ADAM8 associates with PSGL-1 through ezrin-radixin-moesin (ERM) proteins, and that this interaction modulates the expression and function of this adhesion receptor. Results and discussionAssociation of PSGL-1 with ADAM8To identify intracellular molecules able to associate with PSGL-1, we performed a proteomic analysis of HL-60 cell lysates pulled down with the cytoplasmic tail of PSGL-1 fused to GST (GST-PSGL-1cyt) [11]. This analysis revealed the presence of a 98-kDa protein that corresponded to ADAM8 (data not shown). Additional pull-down experiments performed with fragments of the cytoplasmic tail of PSGL-1 fused to GST, detected an additional protein of 75-80 kDa, which likely corresponded to a cleaved form of ADAM8 (Fig. 1A, left panel). To map the region of PSGL-1 involved in this interaction, pull-down experiments from lysates of HL-60 cells were performed with different fragments of the cytoplasmic tail of PSGL-1 fused to GST. We found th...

Regulating inflammation: ADAM8 – a new player in the game

“…These data are in line with a recently published study showing that transgenic mice overexpressing a soluble form of ADAM8 have reduced leukocyte recruitment in an allergeninduced model of lung inflammation [19]. In contrast, knocking down ADAM8 in HL-60 cells significantly increases the number of cells interacting with histamine-activated HUVECs, [5]. As the proteins of the ERM family interact with the cytoplasmic tail of PSGL-1 in the uropod of migrating cells [20,21], it is likely that ADAM8 facilitates migration by cleaving PSGL-1 at the uropod (Fig.…”

“…Taken together, this paper by Dominguez-Luis et al [5] conclusively demonstrates that ADAM8 controls leukocyte-endothelial interaction by shedding PSGL-1. Future studies with ADAM8-deficient mice must elucidate the molecular mechanisms by which ADAM8 influences leukocyte recruitment and inflammatory disease.…”

“…allergen-induced airway inflammation) and osteoclastogenesis [12][13][14]. The study by Dominguez-Luis et al [5] demonstrates that ADAM8 may modulate inflammation by cleaving PSGL-1, which is known to be involved in mediating the first contact between leukocytes and the inflamed endothelium [15], and in selectin-mediated leukocyte activation and recruitment [16][17][18] (Fig. 1B).…”

“…In this issue of the European Journal of Immunology, Dominguez-Luis et al [5] investigate the role of ADAM8 in regulating inflammation. The study demonstrates that ADAM8 is associated with PSGL-1 through ezrin-radixin-moesin (ERM) actin-binding proteins and that this association may increase the proteolytic cleavage of PSGL-1.…”

ADAM-family metalloproteinases in lung inflammation: potential therapeutic targets

“…ADAM8 was found to associate with P-selectin glycoprotein ligand-1 in neutrophilic HL60 cells through ezrin-radixin-moesin actinbinding proteins. Furthermore, ADAM8 caused the proteolytic cleavage of this adhesion receptor and affected leukocyte rolling on activated endothelial cells (31). Moreover, upregulation of ADAM8 on the surface of neutrophils associates with shedding of L-selectin (57,205).…”