Differential Interactions of Thrombospondin-1, -2, and -4 with CD47 and Effects on cGMP Signaling and Ischemic Injury Responses

Michael L. Pendrak,Jeff S. Isenberg,Deane F. Mosher,David D. Roberts,William A. Frazier,Malgorzata Ptaszynska,Douglas S. Annis

doi:10.1074/jbc.m804860200

Abstract

Thrombospondin-1 regulates nitric oxide (NO) signaling in vascular cells via CD47. Because CD47 binding motifs are conserved in the C-terminal signature domains of all five thrombospondins and indirect evidence has implied CD47 interactions with other family members, we compared activities of recombinant signature domains of thrombospondin-1, -2, and -4 to interact with CD47 and modulate cGMP signaling. Signature domains of thrombospondin-2 and -4 were less active than that of thrombospondin-1 for inhibiting binding of radiolabeled signature domain of thrombospondin-1 or SIRPalpha (signal-regulatory protein) to cells expressing CD47. Consistent with this binding selectivity, the signature domain of thrombospondin-1 was more potent than those of thrombospondin-2 or -4 for inhibiting NO-stimulated cGMP synthesis in vascular smooth muscle cells and downstream effects on cell adhesion. In contrast to thrombospondin-1- and CD47-null cells, primary vascular cells from thrombospondin-2-null mice lack enhanced basal and NO-stimulated cGMP signaling. Effects of endogenous thrombospondin-2 on NO/cGMP signaling could be detected only in thrombospondin-1-null cells. Furthermore, tissue survival of ischemic injury and acute recovery of blood flow in thrombospondin-2-nulls resembles that of wild type mice. Therefore, thrombospondin-1 is the dominant regulator of NO/cGMP signaling via CD47, and its limiting role in acute ischemic injury responses is not shared by thrombospondin-2.

Highlights

Nitric oxide (NO) is a major mediator of intracellular and paracellular signal transduction
The signature domain of TSP1 binds with high affinity only to CD47-positive T cells, and its binding is inhibited by a known CD47 blocking antibody
This selectivity correlates with stronger inhibition of NO/cGMP signaling by the signature domain of TSP1 and suggests a more potent activity of TSP1 as opposed to TSP2 or TSP4 for blocking NO-stimulated vascular cell responses and tissue perfusion and survival under ischemic challenge

Summary

Introduction

Nitric oxide (NO) is a major mediator of intracellular and paracellular signal transduction. B and C, wild type or CD47 negative Jurkat cells (106 cells/well) were incubated in PBS plus 0.1% BSA alone or in the presence of TSP1 (10 ␮g/ml) or the CD47 blocking antibody B6H12 (5 ␮g/ml) (B) or an isotype-matched control IgG1 antibody (C), and binding of 125I-SIRP␣ was quantified.

Results

Conclusion