Glycoprotein G;  THBS1;  Thrombospondin;  TSP1

TSP1 is produced by many cell types upon stimulation by cytokines, growth factors, and stress (Isenberg et al. 2009; Rogers et al. 2012). TSP1 is also expressed during early embryogenesis (Bornstein et al. 1990). Treatment with antibodies inhibited blastocyst outgrowth, suggesting a role of TSP1 during early development. In healthy adults, the expression of TSP1 is low but detectable in muscle cells, bone marrow cells, among others (Isenberg et al. 2009). TSP1 expression is regulated at the level of transcription, mRNA stability, translation, and posttranslational modification. Posttranslational modifications have been demonstrated in several domains of this protein and may be important for some TSP1 functions (Roberts and Lau 2011). Expression of TSP1 is elicited by stress, injury, and inflammatory responses (Lopez-Dee et al. 2011). During the acute phase of inflammation, TSP is transiently expressed, and multiple factors seem to modulate the release of TSP1 during this process. Transcription and translation of THBS1 is also induced by hypoxic stress in many cell types, and its induction depends on the transcription factor HIF-2α (Labrousse-Arias et al. 2016). TSP1 is strongly expressed in neutrophils, inducing an intense chemotactic response to injured tissues (Lopez-Dee et al. 2011). Ischemia/reperfusion injury results in a marked elevation of expression of TSP1 and is associated with an increase in cell death (Rogers et al. 2012; Isenberg et al. 2009).

Tissue levels of TSP1 also tend to increase during aging and are associated with the onset of chronic diseases such as atherosclerosis, Alzheimer’s, and type II diabetes (Roberts and Lau 2011; Rogers et al. 2012). Consistent with the latter clinical finding, TSP1 is regulated by glucose. Transcription of TSP1 is activated by the hexosamine pathway of glucose catabolism (Roberts and Lau 2011). Specific inhibitors of glutamine:fructose-6-phosphate amidotransferase, an enzyme controlling the hexosamine pathway, as well as direct inhibitors of protein glycosylation efficiently inhibited glucose-stimulated TSP1 transcription (Roberts and Lau 2011). Hyperglycemia increases TSP1 transcription in kidney mesangial cells by the stimulation of USF2 protein accumulation; this is negatively regulated by an increase in cGMP. Moreover, the regulation of USF2 is mediated by an angiotensin II-dependent mechanism (Visavadiya et al. 2011). TSP1 levels were elevated in adipocytes of rats fed with a high-fat diet to induce obesity and insulin resistance. Expression of TSP1 was increased in adipocytes treated in vitro with high glucose, indicating that TSP1 expression and secretion is modulated by glucose and in models of insulin resistance in vivo and in vitro. Levels of ATP are also responsible for secretion and expression of TSP1 from dendritic cells. This may be regulated by the increase in cAMP, but other mechanisms may be involved (Roberts and Lau 2011). Uptake by receptors is also important for the regulation of extracellular TSP1 levels. Uptake is mediated by the N-terminal domain binding to heparan sulfate proteoglycans and LRP1/calreticulin (Roberts and Lau 2011).

Expression of TSP1 is also altered in malignant cells (Isenberg et al. 2009). For a majority of cancers, expression of TSP1 decreases with tumor progression. Expression of TSP1 is generally higher in the early stages, and its reduction over time is implicated in the angiogenic switch and precedes the increased expression of VEGFA (Isenberg et al. 2009). However, not all cancer types follow this pattern, and higher circulating levels of TSP1 were reported in patients with malignant colorectal carcinoma even though levels in the tumor cells are decreased (Isenberg et al. 2009; Lopez-Dee et al. 2011). Loss of TSP1 in the APC^Min model of colorectal cancer is associated with increased colonic lesions and shorter survival of mice. In this model the absence of TSP1 limited cell death signaling in intestines, which was associated with a systemic deregulation of components of amino acid, energy and lipid metabolism (Soto-Pantoja et al. 2016). Several oncogenes and tumor suppressor genes regulate the transcription or translation of TSP1 including N-Ras, K-Ras, R-Ras,  Myc,  p53, p73, nm23, U19/EAF2, and WT1 (Isenberg et al. 2009). TSP1 is also subject to silencing by hypermethylation in some cancers, and increased expression of TSP1 in human keratinocytes involves DNA hypomethylation.

To study the physiological role of TSP1 in mice, Lawler et al. used homologous recombination to disrupt the mouse Thbs1 gene (Roberts and Lau 2011). Despite its complex regulated expression in embryonic tissues during development, Thbs1 null mice are viable and fertile and do not show gross abnormalities (Lopez-Dee et al. 2011; Roberts and Lau 2011). A reduction in reproductive fitness is associated with altered ovarian follicle morphology and with a deficiency in clearing VEGF by internalizing LRP1 (Roberts and Lau 2011). The original colony of null mice showed in some cases lordotic curvature of the spine and chronic lung inflammation, the latter associated with regulation of latent TGFβ activation. However, rederived Thbs1 null mice in C57Bl/6 background do not exhibit lung inflammation (Roberts and Lau 2011). Thbs1 null mice are leaner than their wild-type counterparts, which is consistent with their increased mitochondrial density and may result from its regulation of mitochondrial biogenesis by modulation of multiple metabolic pathways (Soto-Pantoja et al. 2015). Although TSP1 was originally isolated from and is highly expressed in the α-granules of platelets, the Thbs1 null mice showed no defects in aggregation of washed platelets. However, subsequent studies showed that TSP1 plays a role in platelet function by regulated activity of von Willebrand factor by inhibiting its proteolytic cleavage of ADAM13 (Roberts and Lau 2011). TSP1 also inhibits soluble  guanylate cyclase and cGMP-dependent protein kinase activation in platelets. In the presence of physiological levels of NO or its physiological precursor arginine, TSP1 is necessary for thrombin-induced platelet aggregation, and this activity is mediated through its receptors  CD47 and CD36 (Isenberg et al. 2009; Soto-Pantoja et al. 2015).

The first physiological function identified for TSP1 was inhibition of angiogenesis (Isenberg et al. 2009; Lopez-Dee et al. 2011; Rogers et al. 2012). TSP1 inhibits the growth and migration of endothelial cells and induces apoptosis. TSP1 blocks endothelial cell growth and migration stimulated by pro-angiogenic factors such as fibroblast growth factor-2 ( FGF2) and VEGF in vitro and in vivo (Isenberg et al. 2009). Anti-angiogenic activities are mediated by three TSP1 receptors: CD36, CD148, and  CD47 (Fig. 2). TSP1 binding to CD36 on endothelial cells alters  Src and Fyn kinase activities, with downstream effects on Akt, p38 MAPK, and Syk (Kazerounian et al. 2011) (Fig. 2). TSP1 binding to  CD47 alters heterotrimeric G protein activation, calcium, cGMP, and cAMP signaling in vascular cells (Soto-Pantoja et al. 2015). TSP1 potently inhibits VEGF receptor-2 (VEGFR2) signaling through engaging its receptor  CD47 in endothelial cells, which is dissociated from its constitutive association with VEGFR2 (Kaur et al. 2010). Pro-angiogenic activities of TSP1 have also been reported, which are mediated by the N-terminal domain interacting with α3β1, α4β1, and α9β1 integrins (Fig. 2).

One major function of TSP1 is the regulation of nitric oxide (NO) signaling (Rogers et al. 2012) (Fig. 3). NO is a signaling molecule with several physiological functions. NO is the endothelium-derived relaxing factor that controls arterial smooth muscle tone by relaxing vascular smooth muscle cells (VSMC). NO diffuses rapidly through tissue and binds to its most responsive target soluble guanylate cyclase to stimulate the production of cGMP, which in VSMC triggers their relaxation (Isenberg et al. 2009; Rogers et al. 2012). Endogenous TSP1 functions as a tonic antagonist of NO and signaling in endothelial cells, VSMC, and platelets. The inhibition of NO signaling by TSP1 is mediated primarily by  CD47 and under some conditions by CD36. TSP1 type 1 repeats and synthetic peptides derived from this domain engage CD36, which blocks its ability to uptake myristate required for translocation of  Src kinases to the plasma membrane (Isenberg et al. 2009). TSP1 blocks myristate-stimulated cGMP synthesis in an endothelial NO synthase (eNOS)-dependent manner. This mechanism is associated with the inhibition by TSP1 of angiogenesis and the regulation of endothelial cell chemotaxis. Binding of CD36 is sufficient for the inhibition of NO/cGMP signaling but it is not necessary since TSP1 strongly inhibits NO/cGMP signaling in cells that lack CD36. The C-terminal domain of TSP1 binds  CD47, and this or peptides derived from it potently and redundantly inhibit NO/cGMP signaling at the level of eNOS, soluble guanylate cyclase, and cGMP-dependent protein kinase. Notably, TSP1 also inhibits activation of soluble guanylate cyclase by heme-dependent and heme-independent pharmacological activators (Soto-Pantoja et al. 2015). Thus, these drugs may not be effective for overcoming deficiencies in NO/cGMP signaling in pathological conditions where TSP1 expression is elevated. The expression of  CD47 is also essential for CD36-mediated inhibition of NO signaling. Moreover, a 100-fold lower concentration of TSP1 is sufficient to inhibit NO signaling in vascular cells through  CD47 when compared to CD36 (Isenberg et al. 2009). Thbs1 and   CD47 null mice, but not CD36 null mice, show elevated cGMP levels in vascular cells and muscle tissue.

Open image in new window

The regulation of NO by TSP1 has many physiological functions in the vascular system. Thbs1 null mice are hypersensitive to vasorelaxation induced by NO donors. Furthermore, Thbs1 null mice exhibit decreased central pulse pressure and lower blood pressure than their wild-type counterparts, and these mice have exaggerated hypotensive responses to anesthetics and vasorelaxants (Rogers et al. 2012; Soto-Pantoja et al. 2015). TSP1 also inhibits the acetylcholine-mediated activation of eNOS in the endothelium through  CD47. TSP1 inhibits eNOS activation through the inhibition of calcium signaling and serine-1177 phosphorylation of eNOS. Intravenous treatment with TSP1 and  CD47 antibodies acutely increases blood pressure (Soto-Pantoja et al. 2015). Therefore, TSP1 regulation of NO has vasopressor functions that regulate systemic blood pressure. NO also plays an important role in mediating angiogenic signaling stimulated by VEGF, and TSP1 inhibits angiogenesis in part by inhibiting cGMP synthesis stimulated by VEGF. NO is also an important inhibitor of platelet activation, and TSP1 promotes platelet activation by blocking this activity of NO.

High NO levels can reduce fixed ischemic and ischemia/reperfusion (I/R) injuries by improving blood flow and limiting an inflammatory response. Expression of TSP1 is strongly induced during reperfusion of ischemic cardiac muscle, kidney, and liver tissues in mouse, rat, and porcine I/R injury models (Isenberg et al. 2009). Pretreatment and posttreatment of these injuries decreases I/R injury. This indicates that TSP1 levels are also important in the regulation of tissue perfusion during stress situations.

TSP1 also plays an important role in stem cells (Kaur and Roberts 2016). Primary cells and tissues from TSP1 null mice exhibit elevated self-renewal and stem cell transcription factor expression. These differences are phenocopied in   CD47 null cells and tissues, indicating that  CD47 is the relevant receptor mediating stem cell regulation.   CD47 null cells express higher levels of  Myc, Sox2, Oct4, and Klf4, and treatment of WT but not   CD47 null cells with TSP1 acutely decreases their expression. The increased stem cell numbers in Thbs1 null tissues may contribute to their enhanced regenerative capacity and resistance to stress.

Inflammation is another important physiological response regulated by TSP1 (Soto-Pantoja et al. 2015). TSP1 plays roles in regulating innate and adaptive immunity through its effects on monocytes, macrophages, dendritic cells, T cells, and NK cells. TSP1 is released during acute inflammation and exerts pro- and anti-inflammatory activities in several cell types. This duality can be explained in part by differential regulation of CD36 by peroxisome proliferator-activated receptor (PPAR) and TGFβ (Lopez-Dee et al. 2011). PPAR expression elicits potent anti-inflammatory responses. However, the absence of PPAR in leukocytes causes the increased secretion of TSP1 and stimulates chemotaxis of these cells along with neutrophils (Lopez-Dee et al. 2011). On the other hand, TSP1 activates latent TGFβ by releasing or changing the conformation of its latency-associated protein (Lopez-Dee et al. 2011). TGFβ negatively regulates CD36 which can limit the recruitment of macrophages (Lopez-Dee et al. 2011). Moreover, its interaction with  CD47 is also essential for the recruitment of polymorphonuclear cells. However, in a model of retinal injury, TSP1 limited the migration of microglial cells (Roberts and Lau 2011). Levels of TSP1 regulate migration of phagocytic cells such as macrophages and neutrophils to areas of injury. In an excisional wound-healing model, wound closure in Thbs1 null mice was delayed due to lack of macrophages to the wound. In contrast, lack of TSP1 was protective in liver ischemic injuries (Isenberg et al. 2009). TSP1 also regulates the activation of macrophages (Stein et al., 2016), and M1-differentiated macrophages are increased in tumors that overexpress TSP1 (Isenberg et al. 2009). Furthermore, TSP1 acutely induces superoxide production via NADPH oxidase-2 in macrophages via its binding to α6β1 integrin (Isenberg et al. 2009).

TSP1 is also a potent regulator of T cells. TSP1 stimulates T-cell adhesion, matrix metalloproteinase expression, and migration via α4β1 integrin. Interaction of TSP1 with  CD47 inhibits  CD3-dependent T-cell activation and induces differentiation of CD4⁺ CD25⁺ T cells. Inhibition of T-cell receptor signaling by TSP1 requires a proteoglycan isoform of  CD47 (Soto-Pantoja et al. 2015). TSP1 inhibits the induction of cystathionine β-synthase and cystathionine γ-lyase during T-cell activation, which produce the signaling molecule H₂S (Soto-Pantoja et al. 2015). H₂S is required for optimal activation of T cells, and TSP1 also inhibits T-cell activation induced by exogenous H₂S.

In addition to direct effects on cell signaling, TSP1 can indirectly modulate T-cell activation through extracellular vesicle (EV)/exosome traffic (Fig. 4). Several studies have identified  CD47 on EVs including exosomes. Transfer of CD47⁺ EVs into CD4-deficient T cells resensitizes the cells to TSP1 inhibition. EVs can be internalized by target cells to deliver regulatory microRNAs, and CD47-dependent modulation of signaling in recipient endothelial cells and Jurkat T cells is induced by T-cell-derived EVs (Soto-Pantoja et al., 2015).

Open image in new window

The diverse physiological effects of TSP1 make it an attractive therapeutic target. Since the discovery of the anti-angiogenic properties of this molecule, research has focused on the use of TSP1 analogs as anticancer treatments. ABT-510 is a synthetic nonapeptide derived from the TSR repeats of TSP that interacts with CD36. Preclinical data indicates that ABT-510 inhibits VEGF and  FGF2 signaling; limits endothelial cell migration, proliferation, tube formation, and neovascularization; and induces endothelial and tumor cell apoptosis (Isenberg et al. 2009). ABT-510 inhibits tumor growth in mouse xenograft models. However, ABT-510 therapy did not demonstrate clear clinical efficacy in a clinical trial of patients with stage IV melanoma (Markovic et al. 2007). Further drug optimization or combination with cytotoxic therapy may improve its efficacy.

Blockade of TSP1 signaling through  CD47 may also be beneficial in certain pathologies. Blockade of TSP1 or  CD47 results in improved recovery from ischemia and I/R injuries in several animal models (Isenberg et al. 2009; Rogers et al. 2012). Blocking TSP1 also overcomes the deleterious effects of aging on survival of ischemic stress in a mouse model. Moreover, lack of TSP1 in combination with radiation enhanced tumor growth delay in several syngeneic tumor models (Soto-Pantoja et al. 2015). Blockade of TSP1/ CD47 signaling in cells and wild-type mice simultaneously protected cells, muscle and bone marrow tissue from radiation and confers increased survival of lethal total body irradiation by inducing protective autophagy and stabilizing cell metabolism (Maxhimer et al. 2009; Soto-Pantoja et al. 2015; Miller et al. 2015).

Another emerging therapeutic strategy uses peptide mimics to inhibit TSP1-mediated activation of latent TGFβ (Lu et al. 2016). This TSP1 inhibitor decreased multiple myeloma tumor growth alone and in combination with bortezomib and prevented bone destruction in mouse models.

TSP1 is a multifunctional signaling protein that engages at least ten distinct signaling receptors that are expressed in different combinations on various cell types. Studies using mice lacking TSP1 and several of its receptors have identified physiological functions for TSP1 in regulating platelet hemostasis, angiogenesis, mitochondrial homeostasis, local and systemic regulation of blood flow, neural synapse formation, stress responses, and innate and adaptive immunity. Elevated TSP1 expression contributes to the pathogenesis of acute injuries and several chronic diseases of aging. Conversely, loss of TSP1 expression in some cancers promotes tumor angiogenesis and impairs innate antitumor immunity. Agents targeting specific TSP1 receptors have shown benefit in treating these diseases in animal models and early clinical trials, suggesting that further study of the signaling mechanisms regulated by TSP1 could lead to development of additional therapeutics for these conditions.