Thrombospondin-1 is a CD47-dependent endogenous inhibitor of hydrogen sulfide signaling in T cell activation

Highlights

• Endogenous and exogenous thrombospondin-1 limits the ability of H₂S to enhance T cell receptor-mediated T cell activation.

• CD47 is necessary for thrombospondin-1 to inhibit activation of T cells by H₂S.

• Thrombospondin-1 inhibits H₂S -induced ERK1/2 phosphorylation in activated T cells.

• Thrombospondin-1 limits activation-induced H₂S production by inhibiting expression of cystathionine β-synthase and γ-lyase.

• Thrombospondin-1 signaling through CD47 is the first identified endogenous inhibitor of H₂S signaling.

Abstract

Thrombospondin-1 is a potent suppressor of T cell activation via its receptor CD47. However, the precise mechanism for this inhibition remains unclear. Because H₂S is an endogenous potentiator of T cell activation and is necessary for full T cell activation, we hypothesized that thrombospondin-1 signaling through CD47 inhibits T cell activation by antagonizing H₂S signaling. Primary T cells from thrombospondin-1 null mice were more sensitive to H₂S-dependent activation assessed by proliferation and induction of interleukin-2 and CD69 mRNAs. Exogenous thrombospondin-1 inhibited H₂S responses in wild type and thrombospondin-1 null T cells but enhanced the same responses in CD47 null T cells. Fibronectin, which shares integrin and glycosaminoglycan binding properties with thrombospondin-1 but not CD47 binding, did not inhibit H₂S signaling. A CD47-binding peptide derived from thrombospondin-1 inhibited H₂S-induced activation, whereas two other functional sequences from thrombospondin-1 enhanced H₂S signaling. Therefore, engaging CD47 is necessary and sufficient for thrombospondin-1 to inhibit H₂S-dependent T cell activation. H₂S stimulated T cell activation by potentiating MEK-dependent ERK phosphorylation, and thrombospondin-1 inhibited this signaling in a CD47-dependent manner. Thrombospondin-1 also limited activation-dependent T cell expression of the H₂S biosynthetic enzymes cystathionine β-synthase and cystathionine γ-lyase, thereby limiting the autocrine role of H₂S in T cell activation. Thus, thrombospondin-1 signaling through CD47 is the first identified endogenous inhibitor of H₂S signaling and constitutes a novel mechanism that negatively regulates T cell activation.

Introduction

Thrombospondin-1 (TSP1) is a large (450 kDa) matricellular glycoprotein that plays a pivotal role in regulating vascular homeostasis (Isenberg et al., 2009, Bauer et al., 2010), platelet activation (Isenberg et al., 2008), angiogenesis (Carlson et al., 2008, Miller et al., 2009, Roberts et al., 2012), and immunity (Lopez-Dee et al., 2011). TSP1 mediates these activities by binding to other extracellular matrix components and growth factors, mediating activation of latent TGF-β1 (Schultz-Cherry and Murphy-Ullrich, 1993, Sweetwyne and Murphy-Ullrich, 2012), and binding to at least 12 different cell surface receptors(Murphy-Ullrich and Iozzo, 2012). These receptors include five integrins (Lawler et al., 1988, Chandrasekaran et al., 2000, Calzada et al., 2003, Calzada et al., 2004a, Calzada et al., 2004b, Staniszewska et al., 2007), CD36 (Dawson et al., 1997), CD47 (Gao et al., 1996), CD148 (Takahashi et al., 2012), calreticulin/low density lipoprotein receptor-related protein-1 (LRP1) (Elzie and Murphy-Ullrich, 2004), proteoglycans (Feitsma et al., 2000), and sulfatides (Guo et al., 1992). Among these, TSP1 has the highest affinity for CD47, and this receptor is both necessary and sufficient for TSP1 to inhibit NO-cGMP signaling (Isenberg et al., 2006).

TSP1 regulates T cell activation and function in a domain specific manner. Although TSP1 enhances some T cell actions via its N-terminal domains, such as α4β1 integrin-dependent adhesion and chemotaxis (Li et al., 2002), the dominant effect of soluble TSP1 is the potent inhibition of TCR-mediated T cell activation (Li et al., 2001). This inhibition requires interaction of the C-terminal domain of TSP1 with a proteoglycan isoform of CD47 on the T cell surface (Li et al., 2002, Kaur et al., 2011). The inhibitory activity of TSP1 does not require β1 integrins (Li et al., 2002) and is independent of TGFβ, based on resistance to TGFβ-function blocking antibodies (Li et al., 2001) and the inhibitory activity of a recombinant signature domain of TSP1 that lacks the TGFβ binding and activation sequences in the type 1 repeats (Ramanathan et al., 2011). Further evidence that CD47 ligation is sufficient to inhibit T cell activation derives from the inhibitory activity of some CD47 antibodies and CD47-binding peptides such as 7N3 (FIRVVMYEGKK), but not the corresponding control peptide FIRGGMYEGKK (Li et al., 2001). Despite this evidence that CD47 ligation is necessary and sufficient for inhibiting TCR-dependent T cell activation, the lack of a substantial cytoplasmic domain in CD47 for docking of downstream signaling molecules suggests that lateral interactions with other membrane proteins such as growth factor receptors, integrins, PLIC-1, Fas receptor, and SIRPs are generally required for its signaling functions (reviewed in (Soto-Pantoja et al., 2013).

While the proximal intracellular targets of TSP1/CD47–mediated inhibition of T cell activation are not known, this inhibition occurs downstream of the TCR targeting linker for activated T cells (LAT) and Zap70, but upstream of NFAT activation (Li et al., 2001). TSP1 regulates the activation of soluble guanylate cyclase by NO in Jurkat T lymphoma cells in a calcium-dependent manner (Ramanathan et al., 2011), but this pathway cannot account for the broad effects of CD47 signaling on T cell activation as cGMP signaling is not reported to play a major role in T cell activation and is limited to T cell differentiation (Niedbala et al., 2006).

H₂S is emerging as an important member of the gasotransmitter family that also includes NO and carbon monoxide (CO). At toxic environmental concentrations (> 200 ppm), H₂S inhibits mitochondrial cytochrome c oxidase (Reiffenstein et al., 1992). Lower nontoxic concentrations have physiological functions in neuromodulation ((Abe and Kimura, 1996) and reviewed in (Tan et al., 2009)), metabolic hibernation (Blackstone et al., 2005, Blackstone and Roth, 2007), protection from ischemia/reperfusion injury (Sivarajah et al., 2006, Elrod et al., 2007, Fu et al., 2008, Jha et al., 2008, Tripatara et al., 2008), oxygen sensing (Olson and Whitfield, 2009), vasodilatation (Hosoki et al., 1997, Yang et al., 2008), and promotion of angiogenesis (Wang et al., 2010). Like its gasotransmitter cousins NO and CO, H₂S has transitioned from being perceived exclusively as toxin to recognition that it is an important endogenous signaling molecule. In common with NO, H₂S has been implicated as both a pro- (Bhatia et al., 2005, Collin et al., 2005, Zhang et al., 2007, Cunha et al., 2008) and anti-inflammatory molecule in innate immune cells (Zanardo et al., 2006, Li et al., 2007, Cunha et al., 2008, Sivarajah et al., 2009). Like NO, H₂S relies on its distinctive chemistry for signal transduction, which includes modification of specific protein cysteine residues (termed sulfhydration) and ligation of ferric iron, zinc, or copper centers in metalloproteins (Fukuto et al., 2012).

Recently, we reported that H₂S is a potentiator of T cell activation in primary murine and human T cells and T cell lines (Miller et al., 2012). Exogenously added H₂S, at nanomolar physiological levels (Furne et al., 2008, Shen et al., 2012) enhances both polyclonal and antigen-specific T cell activation. Notably the capacity of T cells to endogenously make H₂S via cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) is turned on as a result of T cell activation. Suppression of CBS and CSE expression by siRNA inhibits T cell activation and T cell proliferation, which can be rescued by supplementation with exogenous H₂S. Therefore, H₂S signaling is a necessary component of T cell activation.

As there are no reported endogenous inhibitors of H₂S signaling, we sought to examine the effect of inhibitory TSP1 signaling through CD47 on H₂S-mediated T cell activation. TSP1 is a logical candidate for regulation of this pathway given its potent and broad T cell inhibitory effects and its potent regulation of NO gasotransmitter signaling.