Abstract
Thrombospondin (TSP) is complexed with transforming growth factor-beta (TGF-beta) in the alpha-granules of stimulated platelets. TSP stripped of associated TGF-beta activity (sTSP) activates latent TGF-beta secreted by bovine aortic endothelial cells (BAE) in culture. To better understand the interactions of TSP with TGF-beta, we investigated which region of sTSP interacts with TGF-beta. The chymotrypsin-resistant core of TSP, which contains the procollagen-like region and the properdin-like type 1 repeats, activated both latent TGF-beta secreted by BAE and a recombinant form of the small latent TGF-beta complex at levels similar to or better than sTSP. The core fragment bound 125I-TGF-beta in solution and shifted the elution profile of 125I-TGF-beta in gel permeation chromatography. Fusion constructs of the type 1, 2, and 3 repeats and the COOH terminus of TSP1 were tested for their ability to activate latent TGF-beta. Only the type 1 construct, containing the three properdin-like repeats of TSP found in the 50-kDa fragment, activated latent TGF-beta. In addition, a polyclonal antibody against the type 1 construct inhibits activation of latent TGF-beta by intact TSP, suggesting that this region is exposed in the intact molecule. These results show that the type 1 properdin-like repeats of TSP are responsible for activating recombinant and endothelial cell-derived latent TGF-beta and that this site is exposed in intact TSP.
Highlights
Thrombospondin (TSP)is complexed with transform- inhibition of endothelial cell growth (5-7), stimulation of ing growth factor-p (TGF-P) in the a-granules of stimu-smooth muscle cell growth in synergy with epidermal growth lated platelets.TSP stripped of associated TGF-j3 activ- factor (EGF) (8), stimulation of fibroblast growth (9),and inhiity(sTSP)activateslatent TGF-P secreted by bovine bition of angiogenesis (10, 11).We have found that TSPl actiaortic endothelial cells (BAE)in culture
For the interchaindisulfide-bonding of trimeric TSPl (19, 20). These results show that the type 1 properdin-like re- The interchaindisulfide bondis followed bythe procollagenpeats of TSP are responsible for activating recombinalnikte domain. This region consists of -90 residues, including 10 and endothelial cell-derived latentTGF-P and that this cysteines, that are homologous to a propeptide that is cleaved site is exposed in intactTSP
Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Cell-Gro, Mediatech,Herndon, VA) supplemented with 4.5 g/liter glucose, 2 mM glutamine, and 20% fetal bovine serum (FBS; HyClone Laboratories, Logan,UT) as described previously (17).normal rat kidney (NRK) cells, clone 49F, (American Q p e Culture Collection, Rockville, MD, CRL 1570)were cultured in DMEM supplemented with 4.5 g/liter glucose,2 mM glutamine, and 10%calf serum (HyClone Laboratories) as described elsewhere (46).All cells tested negative for mycoplasma
Summary
Thrombospondin (TSP)is complexed with transform- inhibition of endothelial cell growth (5-7), stimulation of ing growth factor-p (TGF-P) in the a-granules of stimu-smooth muscle cell growth in synergy with epidermal growth lated platelets.TSP stripped of associated TGF-j3 activ- factor (EGF) (8), stimulation of fibroblast growth (9),and inhiity(sTSP)activateslatent TGF-P secreted by bovine bition of angiogenesis (10, 11).We have found that TSPl actiaortic endothelial cells (BAE)in culture. We showed previously that TSPl interacts with the latent and the active forms of TGF-P (7, 12, 51). To determine which region of TSPl is responsible for the binding to active TGF-P, a solid-phase binding assay was performed using purified TSPl fragments and intact TSP1.
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