Thrombospondin 1

The thrombospondin-1 (TSP1) protein is a member of the Thrombospondin family and is encoded by the gene THBS1. It is a multi-domain matrix glycoprotein that has been shown to be a natural inhibitor of neovascularization and tumorigenesis in healthy tissue. Like the Greek god Janus, both positive and negative modulation of endothelial cell adhesion, motility, and growth have been attributed to TSP1. This should not be surprising considering that TSP1 interacts with at least 12 cell adhesion receptors, including CD36, αv integrins, β1 integrins, syndecan, and integrin-associated protein (IAP or CD47). It also interacts with numerous proteases involved in angiogenesis, including plasminogen, urokinase, matrix metalloproteinase, thrombin, cathepsin, and elastase [Simantov R, Silverstein RL. (2003) CD36: A Critical anti-angiogenic receptor. Frontiers in Bioscience 8:874-882] . Its activity has been mapped to several domains, in particular the amino-terminal heparin-binding domain, the procollagen domain, the properdin-like type I repeats, and the globular carboxy-terminal domain. The protein also contains type II repeats with epidermal growth factor-like homology and type III repeats that contain an RGD sequence [Forslow A, Liu Z, Sundquist KG. (2007) Receptor communication within the lymphocyte plasma membrane: a role for the thrombospondin family of matricellular proteins. Cell. Mol. Life Sci. 64: 66-76] .

The N-terminal heparin-binding domain of TSP1, when isolated as a 25kDa fragment, has been shown to be a potent inducer of cell migration at high concentrations. However, when the heparin-binding domain of TSP1 is cleaved, the remaining anti-angiogenic domains have been shown to have decreased anti-angiogenic activity at low concentrations where increased endothelial cell (EC) migration occurs. This may be explained in part by the ability of the heparin-binding domain to mediate attachment of TSP1 to cells, allowing the other domains to exert their effects. The separate roles that the heparin-binding region of TSP1 plays at high versus low concentrations may be in part responsible for regulating the two-faced nature of TSP1 and giving it a reputation of being both a positive and negative regulator of angiogenesis [Tolsma SS, Volpert OV, Good DJ, Frazier WA, Polverini PJ, Bouck N. (1993) Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity. J Cell Biol 122 (2): 497-511] .

Both the procollagen domain and the type I repeats of TSP1 have been shown to inhibit neovascularization and EC migration. However, it is unlikely that the mechanisms of action of these fragments are the same. The type I repeats of TSP1 are capable of inhibiting EC migration in a Boyden chamber assay after a 3-4 hour exposure, whereas a 36-48 hour exposure period is necessary for inhibition of EC migration with the procollagen domain [Tolsma SS, Volpert OV, Good DJ, Frazier WA, Polverini PJ, Bouck N. (1993) Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity. J Cell Biol 122 (2): 497-511] . Whereas the chorioallantroic membrane (CAM) assay shows the type I repreats of TSP1 to be antiangiogenic, it also shows that the procollagen sequence lacks anti-angiogenic activity. This may be in part because the animo-terminal end of TSP1 differs more than the carboxy-terminal end across species, but may also suggest different mechanisms of action [Iruela-Arispe ML, Lombardo M, Krutzsch HC, Lawler J, Roberts DD. (1999) Inhibition of Angiogenesis by Thrombospondin-1 is mediated by 2 independent regions within the type 1 repeats. Circulation 100: 1423-1431] .

TSP1 contains three type I repeats, only the second two of which have been found to inhibit angiogenesis. The type I repeat motif is more effective than the entire protein at inhibiting angiogenesis and contains not one but two regions of activity. The amino-terminal end contains a tryptophan-rich motif that blocks fibroblast growth factor (FGF-2 or bFGF) driven angiogenesis. This region has also been found to prevent FGF-2 binding ECs, suggesting that its mechanism of action may be to sequester FGF-2. The second region of activity, the CD36 binding region of TSP1, can be found on the carboxy-terminal half of the type I repeats [Iruela-Arispe ML, Lombardo M, Krutzsch HC, Lawler J, Roberts DD. (1999) Inhibition of Angiogenesis by Thrombospondin-1 is mediated by 2 independent regions within the type 1 repeats. Circulation 100: 1423-1431] . It has been suggested that activating the CD36 receptor causes an increase in ECs sensitivity to apoptotic signals [Guo N, Krutzsch HC, Inman JK, Roberts DD. (1997) Thrombospondin 1 and type I repeat peptides of thrombospondin 1 specifically induce apoptosis of endothelial cells. Cancer Res 57:1735–1742] [Sid B, Sartelet H, Bellon G, El Btaouri H, Rath G, Delorme N, Haye B, Martiny L. (2004) Thrombospondin 1: a multifunctional protein implicated in the regulation of tumor growth. Critical Reviews in Oncology/Hematology 49: 245-258] . Type I repeats have also been shown to bind to heparin, fibronectin, TGF-β, and others, potentially antagonizing the effects of these molecules on ECs [Guo N, Zabrenetzky VS, Chandrasekaran L, Sipes JM, Lawler J, Krutzsch HC, Roberts DD. (1998) Differential roles of protein kinase c and pertussis toxin-sensitive g-binding proteins in modulation of melanoma cell proliferation and motility by thrombospondin 1. Cancer Research 58 (14): 3154-3162] . However, CD36 is generally considered to be the dominant inhibitory signaling receptor for TSP1, and EC expression of CD36 is restricted to microvascular ECs [Simantov R, Silverstein RL. (2003) CD36: A Critical anti-angiogenic receptor. Frontiers in Bioscience 8:874-882] .Soluble type I repeats have been shown to decrease EC numbers by inhibiting proliferation and promoting apoptosis. Interestingly, attachment of endothelial cells to fibronectin partially reverses this phenomenon. However this domain is not without a two-faced nature of its own. Bound protein fragments of the type I repeats have been shown to serve as attachment factors for both ECs and melanoma cells [Prater CA, Plotkin J, Jaye D, Frazier WA. (1991) The properdin-like type I repeats of human thrombospondin contain a cell attachment site. J Cell Biol 112(5):1031-40] .

The carboxy-terminal domain of TSP1 is believed to mediate cellular attachment and has been found to bind to another important receptor for TSP1, IAP (or CD47) [Kosfeld MD, Frazier WA. (1992) Identification of active peptide sequences in the carboxyl-terminal cell binding domain of human thrombospondin-1. J Biol Chem 267(23): 16230-6] . This receptor is considered necessary for nitric oxide-stimulated TSP1-mediated vascular cell responses and cGMP signaling [Isenberg JS, Ridnour LA, Dimitry J, Frazier WA, Wink DA, Roberts DD. (2006) CD47 is necessary for inhibition of nitric oxide-stimulated vascular cell responses by thrombospondin-1. J Biol Chem 281(36):26069-80.] . Various domains of and receptors for TSP1 have been shown to have pro-adhesive and chemotactic activities for cancer cells, suggesting that this molecule may have a direct effect on cancer cell biology independent of its anti-angiogenic properties [Chandrasekaran S, Guo NH, Rodrigues RG, Kaiser J, Roberts DD. (1999) Pro-adhesive and chemotactic activities of thrombospondin-1 for breast carcinoma cells are mediated by alpha3beta1 integrin and regulated by insulin-like growth factor-1 and CD98. J Biol Chem 274(16):11408-16] [Taraboletti G, Roberts DD, Liotta LA. (1987) Thrombospondin-induced tumor cell migration: haptotaxis and chemotaxis are mediated by different molecular domains. J Cell Biol 105(5):2409-15] .

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