Regulation of Transforming Growth Factor-β Activation by Discrete Sequences of Thrombospondin 1

Abstract

Transforming growth factor-beta (TGF-beta) is a potent growth regulatory protein secreted by virtually all cells in a latent form. A major mechanism of regulating TGF-beta activity occurs through factors that control the processing of the latent to the biologically active form of the molecule. We have shown previously that thrombospondin 1 (TSP1), a platelet alpha-granule and extracellular matrix protein, activates latent TGF-beta via a protease- and cell-independent mechanism and have localized the TGF-beta binding/activation region to the type 1 repeats of platelet TSP1. We now report that recombinant human TSP1, but not recombinant mouse TSP2, activates latent TGF-beta. Activation was further localized to the unique sequence RFK found between the first and the second type 1 repeats of TSP1 (amino acids 412-415) by the use of synthetic peptides. A peptide with the corresponding sequence in TSP2, RIR, was inactive. In addition, a hexapeptide GGWSHW, based on a sequence present in the type 1 repeats of both TSP1 and TSP2, inhibited the activation of latent TGF-beta by TSP1. This peptide bound to 125I-active TGF-beta and inhibited interactions of TSP1 with latent TGF-beta. TSP2 also inhibited activation of latent TGF-beta by TSP1, presumably by competitively binding to TGF-beta through the WSHW sequence. These studies show that activation of latent TGF-beta is mediated by two sequences present in the type 1 repeats of TSP1, a sequence (GGWSHW) that binds active TGF-beta and potentially orients the TSP molecule and a second sequence (RFK) that activates latent TGF-beta. Peptides based on these sites have potential therapeutic applications for modulation of TGF-beta activation.

Thrombospondin-1 Induces Tyrosine Phosphorylation of Adherens Junction Proteins and Regulates an Endothelial Paracellular Pathway

“…TSP also interacts with other endogenous proteins including TGF␤ (Murphy-Ullrich et al, 1992;Schultz-Cherry and Murphy-Ullrich, 1993), a cytokine associated with multiple biological activities, including wound healing, proliferation, and angiogenesis (Roberts and Sporn, 1993). Activation of latent TGF␤ by TSP has been demonstrated both in vitro (Schultz-Cherry and Murphy-Ullrich, 1993;Schultz-Cherry et al, 1995) and in vivo (Crawford et al, 1998). That TSP activates TGF␤, induces tyrosine phosphorylation and actin reorganization, and influences angiogenesis supports the concept that TSP bioactivity is mediated through changes in EC-EC homophilic adhesion.…”

“…To determine whether TGF␤ might contribute to TSP-induced barrier dysfunction, human recombinant TGF␤ 1 , an anti-TGF␤ antibody (R&D Systems, Minneapolis, MN), as well as a peptide demonstrated to block activation of latent TGF␤ (GGWSHW) (Schultz-Cherry et al, 1995), each were tested in the barrier function assay. To further exclude TGF␤ bioactivity, TSP that was depleted of bound TGF␤, i.e.…”

The effect of thrombospondin-1 on breast cancer metastasis

“…Both of these proteins include the amino acids that comprise ABT-510. These two proteins differed by three amino acid residues, RFK, which has been shown to activate TGF-β [8,[49][50][51]. We found that both of these peptides were able to inhibit tumor growth and inhibit lung metastasis.…”

“…In light of the observation that endogenous TSP-1 promotes metastasis, it is important to ask if treatment with a recombinant version of TSR2 or its related protein TSR2 + RFK promotes metastasis. In TSR + RFK, the sequence has been extended at the N-terminus to include the RFK sequence that has been shown to be essential for the activation for TGF-β [8,[46][47][48][49][50][51][52]. Mice were treated every other day with TSR2 or TSR2 + RFK at 2 mg/kg between days 30 and 90 of age.…”

Blockade of TSP1-Dependent TGF-β Activity Reduces Renal Injury and Proteinuria in a Murine Model of Diabetic Nephropathy

“…25,26 Activation occurs by a nonproteolytic mechanism through binding of the type 1 repeats of TSP1 to a conserved sequence [leucine serine lysine leucine (LSKL)] in the latency associated peptide (LAP) region of the latent complex: LAP binding to the mature domain is required to confer latency, and the RFK sequence of TSP1 disrupts this interaction. 27,28 Peptide mimetics of these sequences can be used to antagonize TSP1-mediated latent TGF-␤ activation (LSKL and GGWSHW) or, alternatively, to stimulate activation [lysine arginine phenylalanine lysine (KRFK) and RKPK]. 29 Many factors associated with increased TGF-␤ activity in diabetes also stimulate TSP1 expression.…”