Thrombospondin 2 Potentiates Notch3/Jagged1 Signaling

He Meng,Xiaojie Zhang,Kurt D Hankenson,Michael M Wang

doi:10.1074/jbc.m803650200

He Meng, Xiaojie Zhang + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m803650200

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Abstract

Extracellular thrombospondins (TSP or THBS) and the Notch family of transmembrane receptors share a role in multiple, overlapping cellular functions and participate in developmental signaling and pathological reactions to tissue injury. We demonstrate that TSP2, but not TSP1, enhances the potency of Notch3 signal transduction. In addition, TSP2 reduces cancer cell proliferation in a Notch-ligand dependent fashion. The loss of TSP2 in knock-out mice reduces Notch target gene expression. TSP2 binds directly to Notch3 and Jagged1. TSP1 also binds to Notch3 and Jagged1; however, only TSP2 augments the interaction between Notch3 and Jagged1. These studies demonstrate that the diverse functions of TSP2 may also include a role as an intermediary protein that facilitates transcellular receptor-ligand interactions.

Highlights

The Notch signaling pathway is an evolutionarily conserved, intercellular signaling system that plays an essential role in development of multiple organ systems [8]
We investigated the functional interaction between Thrombospondin 2 (TSP2) and Notch3; TSP2 was chosen for analysis because of our interest in Notch3 signal modification in vascular smooth muscle, which synthesizes TSP2
Our experiments demonstrate for the first time that the matricellular protein TSP2 modifies Notch signaling potency and binds to Notch3 and Jagged1

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—293A (Qbiogene), L fibroblast lines, and A2780 were propagated in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). Binding Assays—The following purified recombinant proteins were obtained from R&D Systems: recombinant human Notch-3-Fc fusion (first 11 EGF repeats), rat Jagged1-Fc, human full-length TSP1 and TSP2, and control human IgG1 Fc. Proteins (5 ␮g) were labeled with Alexa700-succinimide (10 ␮g) in PBS at room temperature for 1 h. The level of Notch activation, H460 cells were transfected with Hes1-luciferase [30]. Coculture with ligand-expressing cells significantly increased Hes1-luciferase activity in. Ligand-stimulated Hes1-luciferase reporter activity was significantly amplified with recombinant TSP2 protein, demonstrating that the stim-. Alexa-labeled ligands have been used before in binding studies ulatory effect of TSP2 does not require co-expression within the of proteins adsorbed to plastic [28]. Of Notch (on the plate or in solution) are bound by Fc and BSA TSP2 can activate multiple intracellular signaling pathways control proteins.

RESULTS

Cell lysate

DISCUSSION