Rusnati M, Taraboletti G, Urbinati C, Tulipano G, Giuliani R, Molinari-Tosatti M P, Sennino B, Giacca M, Tyagi M, Albini A, Noonan D, Giavazzi R, Presta M
Chair of General Pathology and Immunology, University of Brescia, Brescia, Italy.
FASEB J. 2000 Oct;14(13):1917-30. doi: 10.1096/fj.99-0902com.
Tat protein, a trans-activating factor of the human immunodeficiency virus type 1, acts also as an extracellular molecule modulating gene expression, cell survival, growth, transformation, and angiogenesis. Here we demonstrate that human thrombospondin-1 (TSP), a plasma glycoprotein and constituent of the extracellular matrix, binds to glutathione-S-transferase (GST)-Tat protein but not to GST. Scatchard plot analysis of the binding of free GST-Tat to immobilized TSP reveals a high-affinity interaction (Kd equal to 25 nM). Accordingly, TSP inhibits cell internalization and HIV-1 LTR trans-activating activity of extracellular Tat in HL3T1 cells with ID50 equal to 10-30 nM. Also, TSP inhibits cell interaction and mitogenic activity of extracellular Tat in T53 Tat-less cells. TSP is instead ineffective when administered after the interaction of Tat with cell surface heparan-sulfate proteoglycans has occurred, in keeping with its ability to prevent but not disrupt Tat/heparin interaction in vitro. Finally, TSP inhibits the autocrine loop of stimulation exerted by endogenous Tat in parental T53 cells. Accordingly, TSP overexpression inhibits cell proliferation, angiogenic activity, and tumorigenic capacity of stable T53 transfectants. Our data demonstrate the ability of TSP to bind to Tat protein and to affect its LTR trans-activating, mitogenic, angiogenic, and tumorigenic activity. These findings suggest that TSP may be implicated in the progression of AIDS and in AIDS-associated pathologies by modulating the bioavailability and biological activity of extracellular Tat.
Tat蛋白是人类免疫缺陷病毒1型的反式激活因子，它还作为一种细胞外分子调节基因表达、细胞存活、生长、转化和血管生成。在此我们证明，人血小板反应蛋白-1（TSP），一种血浆糖蛋白和细胞外基质的组成成分，能与谷胱甘肽-S-转移酶（GST）-Tat蛋白结合，但不与GST结合。对游离GST-Tat与固定化TSP结合的Scatchard图分析显示存在高亲和力相互作用（解离常数Kd等于25 nM）。因此，TSP抑制HL3T1细胞中细胞内化以及细胞外Tat的HIV-1长末端重复序列（LTR）反式激活活性，半数抑制浓度（ID50）等于10 - 30 nM。此外，TSP抑制T53无Tat细胞中细胞外Tat的细胞相互作用和促有丝分裂活性。相反，当Tat与细胞表面硫酸乙酰肝素蛋白聚糖相互作用后再给予TSP则无效，这与其在体外防止但不破坏Tat/肝素相互作用的能力一致。最后，TSP抑制亲本T53细胞中内源性Tat施加的自分泌刺激环。因此，TSP过表达抑制稳定的T53转染子的细胞增殖、血管生成活性和致瘤能力。我们的数据证明了TSP与Tat蛋白结合并影响其LTR反式激活、促有丝分裂、血管生成和致瘤活性的能力。这些发现表明，TSP可能通过调节细胞外Tat的生物利用度和生物学活性而参与艾滋病的进展以及与艾滋病相关的病理过程。
