Activation of latent TGF-β by thrombospondin-1: mechanisms and physiology
Introduction
TGF-β is a potent regulatory protein with effects on growth and differentiation and gene expression [1], [2], [3]. Appropriate levels of TGF-β activity are essential for the well being of the organism. Lack of sufficient TGF-β can result in immunological and inflammatory disturbances, developmental abnormalities, deficient wound healing, and increased tumorigenesis [4], [5]. Conversely, excessive TGF-β activity leads to scarring, the development of fibrotic diseases in multiple organ systems, immune suppression, and possibly, enhancement of the later stages of tumor progression [6], [7], [8]. TGF-β is synthesized and secreted by most cell types as an inactive precursor complex, termed latent TGF-β. Since most cell types express TGF-β and its signaling receptors, a major determinant of the level of TGF-β activity, present in a particular tissue during a specific physiologic or pathologic event, occurs at the level of processes that regulate activation of the latent complex. In this review, we will briefly discuss the structure of the latent complex and physiologic regulation of the activation process. There are other recent reviews that deal with activation in a comprehensive manner [9]. Therefore, this review will be limited to a discussion of our current understanding of how the secreted and extracellular matrix protein, thrombospondin-1 (TSP), regulates latent TGF-β activation.
Section snippets
The latent TGF-β complex
There are three mammalian isoforms of TGF-β (1, 2, and 3): each is secreted by cells as a disulfide linked homodimer consisting of a 278 amino acid pro-peptide, known as the latency associated peptide (LAP), that is non-covalently associated with a 112 amino acid peptide containing the bioactive domain (Fig. 1). The LAP domain is cleaved intracellularly from the mature region by an endoprotease furin [10]; however, the LAP and mature regions remain associated with each other — purportedly
Physiologic mechanisms of activation
For TGF-β to be able to signal through its receptors, it must be converted to the active state. In vitro, latent TGF-β can be activated by a number of denaturing agents, including heat, extremes of pH, chaotropic agents, and detergents. The physiologic mechanisms that regulate latent TGF-β activation are not well understood. Proteolysis by plasmin, cathepsin, and other enzymes can activate latent TGF-β [15], [16]. There is evidence from endothelial cell-smooth muscle cell co-culture systems
Thrombospondin: an important modulator of matrix structure and cellular function
Thrombospondin (TSP) is a multifunctional protein that exists as both a secreted protein and as an insoluble extracellular matrix molecule (reviewed in Refs. [28], [29], [30]). TSP1, the best-characterized member of the family of five thrombospondin isoforms, is a major component of platelet α-granules. TSP1 acts as an immediate early response gene, being rapidly up-regulated in response to serum and growth factors such as TGF-β and PDGF. Its promoter region contains AP-1, AP-2, NFκB, and SRE
Thrombospondin activates TGF-β by direct interactions with latent TGF-β
We observed that human platelet TSP1 caused growth inhibition of aortic endothelial cells at least partially via a TGF-β sensitive mechanism. This led to the finding that TSP is complexed with active TGF-β in platelet releasates, conditioned medium, and serum [38]. The TGF-β associated with TSP is in the biologically active form. It is estimated that 1 in every 500 TSP-1 molecules purified from platelet α-granules has TGF-β activity associated with it. Subsequently, we showed that exogenous TSP
Identification of TGF-β binding sites in thrombospondin
We used proteolytic fragments, recombinant fragments, and peptide approaches to localize two sites in TSP that interact with the latent TGF-β complex and to determine the site in TSP-1 responsible for activation. We have localized the active site of TSP to the three type 1 properdin-like repeats of the molecule [45]. A three amino acid sequence (RFK) is located between the first and second type 1 repeats [46]. When expressed as a peptide, the RFK sequence is functional at picomolar
The TSP binding site in the LAP
As anti-peptide antibodies to the N-terminus of the LAP block TSP-dependent activation of latent TGF-β and the activating RFK sequence is involved in TSP interactions with the LAP, we proposed that LAP-TSP interactions through the RFK site in TSP-1 are important for activation. In order to better characterize TSP-LAP interactions, we sought to identify a site in the LAP complementary to the (K)RFK sequence in TSP-1. Using the Molecular Recognition Theory as a starting point [47], we identified
Evidence for a role for thrombospondin-dependent activation under physiologic conditions
In an attempt to understand the physiologic and pathologic conditions under which thrombospondin-dependent mechanisms of latent TGF-β activation might be operative, we have turned to both in vitro and in vivo systems. Physiologic examples of TSP-regulated activation of latent TGF-β include: dermal wound healing studies in TSP-1 null mice; the role of TSP-1 regulated TGF-β activation in post-natal development; and bleomycin-induced pulmonary fibrosis in rats. Studies are currently ongoing to
TSP-dependent TGF-β regulation in bleomycin-induced pulmonary fibrosis
In a rat model of bleomycin-induced pulmonary fibrosis which mimics human idiopathic pulmonary fibrosis, infiltrating macrophages secrete active TGF-β with temporal characteristics that correspond to increased expression of TSP1 protein by these cells. The development of fibrotic disease in both these animals is dependent upon TGF-β mediated processes [51]. In collaboration with Dr Nasreen Khalil, we observed that anti-TSP antibodies block the stimulation of TGF-β activity. Alveolar
Issues and future directions
There are many issues that remain to be addressed regarding: (a) the mechanism of activation of latent TGF-β by thrombospondin-1; and (b) determination of the physiologic and pathologic situations in which this activation mechanism is operative. The working hypothesis is that TSP-binding to the latent complex induces a conformational change in the LAP such that the receptor-binding sites near the C-terminus of the mature domain are now accessible to TGF-β receptors. TSP bound to LAP may impose
Summary
We have shown in a variety of cultured cell systems and under both physiologic and certain pathologic situations in vivo that TSP plays a significant role in regulating the activation of latent TGF-β. Based on the ability of the KRFK-activating peptide and of the LSKL or GGWSHW inhibitory peptides to either substitute or to block endogenous TSP action, respectively, we suggest that direct interactions of TSP with latent TGF-β represents a major mechanism for TSP-mediated activation of latent
Acknowledgements
The work discussed in this review was supported by a grant from the American Cancer Society (CB-78) and by NIH Grants HL50061, HL54624 to JEMU. The authors would like to acknowledge the essential contributions of Stacey Schultz-Cherry, Solange Ribeiro, Antonio Pallero, Yun Su, David Roberts, Larry Gentry, Deane Mosher, Sue Crawford, Veronica Stellmach, Noel Bouck, Jack Lawler, Peter Polverini, Nasreen Khalil, Christian Hugo, Ed Blalock, and the members of these laboratories to these studies.
References (63)
- et al.
Latent transforming growth factor-β: structural features and mechanisms of action
Kid. Int.
(1997) - et al.
Photodynamic therapy inhibits transforming growth factor-β activity associated with vascular smooth muscle cell injury
J. Vasc. Surg.
(1997) - et al.
The integrin αvβ6 binds and activates latent TFG-β1: a mechanism for regulating pulmonary inflammation and fibrosis
Cell
(1999) Thrombospondins
Curr. Opin. Cell Biol.
(1991)- et al.
Activation of alveolar macrophage-derived latent TGF-β1 by plasmin requires interaction with TSP-1 and the TSP-1 cell surface receptor CD-36
Amer. J. Pathol.
(1999) - et al.
Regulation of transforming growth factor-β activation by discrete sequences of thrombospondin 1
J. Biol. Chem.
(1995) - et al.
Thrombospondin-1 is a major activator of TGF-β1 in vivo
Cell
(1998) - et al.
Thrombospondin1 is expressed by proliferating mesangial cells and is up-regulated by PDGF and bFGF in vivo
Kidney Int.
(1995) - et al.
Thrombospondin peptides are potent inhibitors of mesangial and glomerular endothelial cell proliferation in vitro and in vivo in experimental mesangial proliferative glomerulonephritis in the rat
Kidney Int.
(1999) - et al.
Identification of glucose-regulated genes in human mesangial cells by mRNA differential display
Biochem. Biophys. Res. Comm.
(1997)