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

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 SIRP␣ (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.Nitric oxide (NO) is a major mediator of intracellular and paracellular signal transduction. NO preserves vascular health by minimizing the adhesion of inflammatory cells to the vessel wall, limiting platelet activation, and increasing blood vessel diameter and blood flow by relaxing vascular smooth muscle cells (VSMC).3 These actions of NO are mediated by activating soluble isoforms of guanylate cyclase (sGC) to increase cGMP levels, resulting in downstream activation of cGMP-dependent protein kinases and ion channels (1).Physiological NO/cGMP signaling is limited by several phosphodiesterases that degrade cGMP and by thrombospondin-1 (TSP). TSP1 is a secreted protein that is produced by vascular and inflammatory cells that regulates cellular behavior by engaging several cell surface receptors. Recently we reported that TSP1 potently blocks NO-stimulated prosurvival responses in endothelial and VSMC (2, 3). TSP1 also plays a role in promoting platelet thrombus formation and hemostasis by antagonizing the antithrombotic activity of NO (4). In all of these vascular cells, picomolar concentrations of TSP1 are sufficient to block NO-stimulated fluxes in cGMP by engaging its receptor CD47 (5). Nanomolar concentrations of TSP1 further inhibit the same signaling pathway by inhibiting CD36-mediated uptake of myristate into vascular cells (6). In vivo, mice lacking TSP1 demonstrate elevated basal tissue cGMP levels and greater increases in regional blood flow in response to a NO challenge than wild type controls (4). After an ischemic insult, the absence of TSP1 o...

Regulation of Cellular Redox Signaling by Matricellular Proteins in Vascular Biology, Immunology, and Cancer

“…This suggested that TSP2 and other TSPs may also inhibit NO signaling via interaction with CD47. A comparison of recombinant signature domains of TSP1, TSP2, and TSP4 showed moderate competition between the signature domains of TSP2 and TSP1 for binding to cells expressing CD47 but no significant inhibition by the signature domain of TSP4 (84). Consistent with these data, the three paralogs showed decreasing potency for inhibiting NO-stimulated cGMP accumulation in VSMCs in the order TSP1>TSP2>TSP4.…”

“…In the absence of exogenous TSP1, ligation of CD47 by the CD47 antibodies C1Km1 and B6H12 also inhibited NO signaling. This inhibitory activity of B6H12 is notable because B6H12 inhibits TSP1 binding to CD47 (84), suggesting that this antibody can, under TSP1-depleted conditions, act as a mimic of TSP1 and activate CD47 signaling to inhibit NO stimulation of sGC independent of its ability to prevent TSP1 binding to CD47.…”

Signal regulatory protein alpha (SIRPα)/CD47 interaction and function

“…One puzzling aspect concerning CD47 is that much of the data on thrombospondin binding is from peptide binding analysis but the structure of the relevant thrombospondin domain shows that the peptides are in the core of the domain and not readily accessible [11]. This domain shows no affinity for the extracellular IgSF domain of CD47 [12] but direct binding of comparable proteins to CD47 at the cell surface has recently been shown [13] which might indicate that other parts of CD47 are involved. CD47 interactions and signalling are complex and beyond detailed analysis in this review.…”

Cellular, Pharmacological, and Biophysical Evaluation of Explanted Lungs from a Patient with Sickle Cell Disease and Severe Pulmonary Arterial Hypertension

“…Treating PASMCs with exogenous TSP1 (2.2 nmol/L, 24 hours) increased ET-1, ETA, and CD47 protein expression. Conversely, a CD47 antibody (clone B6H12, 1 μg/mL) that blocks TSP1 binding 34 inhibited expression of TSP1, ET-1, and ETA in SCD-associated PASMCs (Fig. 1B).…”