Cell surface nucleolin is a novel ADAMTS5 receptor mediating endothelial cell apoptosis

Abstract

A Disintegrin and Metalloproteinase with ThromboSpondin motif (ADAMTS) 5 functions as an anti-angiogenic and anti-cancer protein independent of its metalloproteinase activity. Both full-length ADAMTS5 and TS5-p45, the autocatalytically cleaved C-terminal 45 kDa truncate of ADAMTS5, inhibits angiogenesis, and induces endothelial cell (EC) apoptosis. However, how ADAMTS5 triggers EC apoptosis remains unclear. This work shows that caspase-8 (Cas-8) and caspase-9 (Cas-9) are involved in TS5-p45-induced EC apoptosis. We identify cell surface nucleolin (NCL) as a novel high-affinity receptor for TS5-p45 in ECs, mediating TS5-p45’s cell surface binding and pro-apoptotic function. We show that the central RNA-binding domain (RBD) of NCL is essential and sufficient for its binding to TS5-p45. Upon interacting with EC surface NCL, TS5-p45 is internalized through clathrin- and caveolin-dependent endocytosis and trafficked to the nucleus via late endosomes (LEs). We demonstrate that the nuclear trafficking of TS5-p45 is important for its pro-apoptotic activity as disruption of LE membrane integrity with an endosomolytic peptide suppressed both nuclear trafficking and pro-apoptotic activity of TS5-p45. Through cell surface biotinylation, we revealed that cell surface NCL shuttles extracellular TS5-p45 to the nucleus to mediate apoptosis. Furthermore, blocking the importin α1/ß1 receptor hindered the nuclear trafficking of TS5-p45, suggesting the involvement of the nuclear importing machinery for this nuclear translocation. RNA-seq identified many apoptosis-related genes that are differentially expressed at least two-fold in TS5-p45-treated ECs, with 10 of them qRT-PCR-validated and at least 5 of these genes potentially contributing to TS5-p45-NCL-induced apoptosis. Altogether, our work identifies NCL as a novel cell surface receptor for ADAMTS5 and demonstrates the critical role of NCL-mediated internalization and nuclear trafficking for ADAMTS5-induced EC apoptosis. These findings reveal novel mechanistic insights of the secreted metalloproteinase ADAMTS5 in angiogenesis inhibition.

Limb expression 1‐like protein promotes epithelial–mesenchymal transition and epidermal growth factor receptor‐tyrosine kinase inhibitor resistance via nucleolin‐mediated ribosomal RNA synthesis in non‐small cell lung cancer

“…In contrast, NCL protein contains an N-terminal, an RBD, and a C-GAR domain (Figure 5L). 40 Our results suggested that the RBD is sufficient for NCL to bind LIX1L (Figure 5M-R; Figure S4K-P). Furthermore, we confirmed the association between LIX1L-RBD and NCL-RBD and found that the association was blocked by RNase A (Figure 5S-U; Figure S4Q,R,U), indicating that RNA may mediate the association.…”

Uncovering the ligandome of low-density lipoprotein receptor-related protein 1 in cartilage: a top-down approach to identify therapeutic targets

“…In agreement with these studies, we found pro-cell survival role of LRP1 in human articular chondrocytes. Furthermore, several studies demonstrated cell survival and death functions for both previously reported and newly identified LRP1 ligands such as TIMP3 [50][51][52][53][54][55], ADAMTS5 [56], CTGF [57,58], CEMIP [59], SLIT2 [60,61], IGFBP7 [62][63][64] and HMGB2 [65]. The mechanisms by which LRP1 ligands regulate cell survival in chondrocytes remain unclear but the prolonged presence of one or several LRP1 ligands in the extracellular milieu may gradually alter cellular environment and metabolism leading to cell death.…”

Nucleolin‑based targeting strategies in cancer treatment: Focus on cancer immunotherapy (Review)

“…By binding with Fas ( 70 ), or with Ras via the C-terminal domain ( 71 ), NCL significantly promotes cancer cell proliferation and inhibits apoptosis by decreasing the expression of the pro-apoptotic BAX gene ( 72 ). Targeting cell-surface NCL might trigger multiple inhibitory effects, depending on the cell type ( 68 , 73 ). Taken together, to date, these observations highlight a critical role for NCL in tumorigenesis and tumor progression.…”