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Abstract
Transforming growth factor-beta (TGF-beta) is secreted as a latent complex of the latency-associated peptide (LAP) and the mature domain, which must be activated for TGF-beta to signal. We previously identified thrombospondin 1 (TSP1) as a physiologic activator of TGF-beta in vitro and in vivo. The WSXW sequences in the type 1 repeats of TSP1 interact with the mature domain of TGF-beta, and WSXW peptides inhibit TSP1-mediated activation by blocking TSP1 binding to the TGF-beta latent complex. However, the binding site for the WSXW sequence was not identified. In this report, we show that the WSXW sequences bind the (61)VLAL sequence in mature TGF-beta and also bind (77)VLAL in LAP. A glutathione S-transferase (GST) fusion protein of the second TSP1 type 1 repeat (GST-TSR2) binds immobilized VLAL peptide. VLAL peptides inhibit binding of LAP and mature TGF-beta to soluble GST-TSR2 and immobilized WSXW peptide. VLAL peptide inhibits TSP1-mediated activation of recombinant and endothelial cell-derived latent TGF-beta. Furthermore, TGF-beta or LAP deleted in the VLAL sequence fails to bind immobilized WSXW or soluble GST-TSR2, indicating that binding to both VLAL sequences is important for association of TSP1 and the latent complex. Additionally, TSP1 is unable to activate latent TGF-beta when VLAL is deleted from the mature domain. These data show that the WSXW motif binds VLAL on both LAP and mature TGF-beta, and these interactions are critical for TSP1-mediated activation of the TGF-beta latent complex.
Highlights
Transforming growth factor- (TGF-)1 is a cytokine involved in diverse processes, including development, angiogenesis, wound healing, inflammation, neoplasia, and fibrosis [1,2,3,4]
The WSXW sequences in the type 1 repeats of thrombospondin 1 (TSP1) interact with the mature domain of TGF-, and WSXW peptides inhibit TSP1-mediated activation by blocking TSP1 binding to the TGF- latent complex
We show that the WSXW sequences bind the 61VLAL sequence in mature TGF- and bind 77VLAL in latency-associated peptide (LAP)
Summary
Cell Culture—Cells were cultured in Dulbecco’s modified Eagle’s medium containing 4.5 g/liter glucose, 2 mM glutamine, and 10% fetal bovine serum. Mink lung epithelial cells (Mv1Lu) transfected with the TGF- response element of the human plasminogen activator inhibitor-1 gene promoter fused to the firefly luciferase reporter gene were a generous gift from Dr Daniel Rifkin (New York University) [24] and were cultured in the above medium supplemented with 200 g/ml G-418 (Invitrogen). The purified PCR product was inserted, according to the manufacturer’s instructions, into the mammalian expression vector pEF6/V5-His-TOPO (Invitrogen). LAP-FLAG protein expression in transfected COS-1 cells was confirmed by immunoblot, and the ability to confer latency to active TGF- was verified by the PAIL assay. Transfection and Conditioned Medium Collection—Upon reaching 70% confluence in 100-mm dishes, COS-1 cells were washed in serumfree medium and transfected with 2 g of DNA and 6 l of Fugene-6 (Roche Applied Science) per the manufacturer’s instructions in a final volume of 5 ml of serum-free Dulbecco’s modified Eagle’s medium with ITS (insulin, transferrin, selenium) supplement (Sigma).
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