Thiolutin is a sulfur-based microbial compound with known activity as an angiogenesis inhibitor. Relative to previously studied angiogenesis inhibitors, thiolutin is a remarkably potent inducer of heat shock protein 27 (Hsp27) phosphorylation. This phosphorylation requires p38 kinase but is independent of increased p38 phosphorylation. To elucidate how thiolutin regulates Hsp27 phosphorylation and ultimately angiogenesis, Hsp27 was immunoprecipitated using non-phosphorylated protein and phospho-Ser78 specific antibodies from lysates of thiolutin treated and untreated human umbilical vein endothelial cells and analyzed by LC-MS. LC-MS analyses of proteolytic digests provided 100% sequence coverage of Hsp27. Analysis revealed a novel post-translational modification of Hsp27 involving truncation of the N-terminal Met and acetylation of the penultimate Thr. Analysis of a Glu-C fragment containing two phosphorylation sites, Ser78 and Ser82, and a tryptic fragment containing the other phosphorylation site, Ser15, enabled quantitative analysis of Hsp27 phosphorylation by LC-MS. The strategy revealed details of Hsp27 phosphorylation, including significant double phosphorylation at Ser78 and Ser82, that would be difficult to obtain by traditional approaches because oligomerization of the hydrophobic N-terminal region of the molecule prevents efficient enzymatic cleavage. The combination of Western blotting, immunoprecipitation, and LC-MS provides a quantitative analysis of thiolutin-stimulated Hsp27 phosphorylation and further defines the role of Hsp27 in the anti-angiogenic activities of thiolutin and related dithiolethiones. Human plasma fibronectin binds with high affinity to the inflammation-induced secreted protein TSG-6. Fibronectin binds to the CUB_C domain of TSG-6 but not to its Link module. TSG-6 can thus act as a bridging molecule to facilitate fibronectin association with the TSG-6 Link module ligand thrombospondin-1. Fibronectin binding to TSG-6 is divalent cation-independent and is conserved in cellular fibronectins. Based on competition binding studies using recombinant and proteolytic fragments of fibronectin, TSG-6 binding localizes to type III repeats 9-14 of fibronectin. This region of fibronectin contains the Arg-Gly-Asp sequence recognized by 1 integrin, but deletion of that sequence does not prevent TSG-6 binding, and TSG-6 does not inhibit cell adhesion on fibronectin substrates mediated by this integrin. This region of fibronectin is also involved in fibronectin matrix assembly, and addition of TSG-6 enhances exogenous and endogenous fibronectin matrix assembly by human fibroblasts. Therefore, TSG-6 is a high affinity ligand that can mediate fibronectin interactions with other matrix components and modulate some interactions of fibronectin with cells. Inhibition of tumor growth by thrombospondin-1 (TSP1) is generally attributed to its anti-angiogenic activity, but effects on tumor immunity should also be considered. We show that over-expression of TSP1 in melanoma cells increases macrophage recruitment into xenograft tumors grown in nude or beige/nude mice. In vitro, TSP1 acutely induces expression of plasminogen activator inhibitor-1 (PAI-1) by monocytic cells, suggesting that TSP1-induced macrophage recruitment is at least partially mediated by PAI-1. Tumor-associated macrophages can either promote or limit tumor progression. The percentage of M1 polarized macrophages expressing inducible nitric oxide synthase is increased in TSP1-expressing tumors. Furthermore, soluble TSP1 stimulates killing of breast carcinoma and melanoma cells by interferon-γ-differentiated U937 cells in vitro via release of reactive oxygen species. TSP1 causes a significant increase in phorbol ester-mediated superoxide generation from differentiated monocytes by interaction with alpha6beta1 integrin through its N-terminal region. The N-terminal domain of thrombospondin-2 also stimulates monocyte superoxide production. Extracellular calcium is required for the TSP1-induced macrophage respiratory burst. Thus, TSP1 may play an important role in anti-tumor immunity by enhancing recruitment and activation of M1 tumor-associated macrophages, which provides an additional selective pressure for loss of TSP1 and thrombospondin-2 expression during tumor progression.
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