Growth factors such as thrombin and transforming growth factor (TGF)-β facilitate glycosaminoglycan (GAG) chain hyperelongation on proteoglycans, a phenomenon that increases lipoprotein binding in the vessel wall and the development of atherosclerosis. TGF-β signals via canonical carboxy terminal phosphorylation of R-Smads and also non-canonical linker region phosphorylation of R-Smads. The G protein coupled receptor agonist, thrombin, can transactivate the TGF-β receptor leading to both canonical and non-canonical Smad signalling. Linker region phosphorylation drives the expression of genes for the synthesis of the proteoglycan, biglycan. Proteoglycan synthesis involves core protein synthesis, the initiation of GAG chains and the subsequent elongation of GAG chains. We have explored the relationship between the thrombin stimulated phosphorylation of individual serine and threonine sites in the linker region of Smad2 and the expression of GAG initiation xylosyltransferase-1 (XT-1) and GAG elongation chondroitin 4-sulfotransferase-1 (C4ST-1) and chondroitin synthase-1 (CHSY-1) genes. Thrombin stimulated the phosphorylation of all four target residues (Thr220, Ser245, Ser250 and Ser255 residues) with a similar temporal pattern - phosphorylation was maximal at 15 min (the earliest time point studied) and the level of the phospho-proteins declined thereafter over the following 4 h. Jnk, p38 and PI3K, selectively mediated the phosphorylation of the Thr220 residue whereas the serine residues were variously phosphorylated by multiple kinases. Thrombin stimulated the expression of all three genes - XT-1, C4ST-1 and CHSY-1. The three pathways mediating Thr220 phosphorylation were also involved in the expression of XT-1. The target pathways (excluding Jnk) were involved in the expression of the GAG elongation genes (C4ST-1 and CHSY-1). These findings support the contention that individual Smad linker region phosphorylation sites are linked to the expression of genes for the initiation and elongation of GAG chains on proteoglycans. The context of this work is that a specific inhibitor of GAG elongation represents a potential therapeutic agent for preventing GAG elongation and lipid binding and the results indicate that the specificity of the pathways is such that it might be therapeutically feasible to specifically target GAG elongation without interfering with other physiological processes with which proteoglycans are involved.

中文翻译：

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单个Smad2接头区域的磷酸化位点决定了蛋白聚糖和糖胺聚糖合成基因的表达。

诸如凝血酶和转化生长因子（TGF）-β之类的生长因子促进蛋白聚糖上糖胺聚糖（GAG）链的过度伸长，这种现象增加了血管壁中脂蛋白的结合以及动脉粥样硬化的发展。TGF-β通过R-Smads的标准羧基末端磷酸化以及R-Smads的非典型接头区域磷酸化发出信号。G蛋白偶联受体激动剂凝血酶可激活TGF-β受体，从而导致规范性和非规范性Smad信号传导。接头区域的磷酸化驱动蛋白聚糖（双糖链蛋白聚糖）合成基因的表达。蛋白聚糖合成涉及核心蛋白合成，GAG链的起始以及随后GAG链的延长。我们已经探索了凝血酶刺激的Smad2接头区域中的单个丝氨酸和苏氨酸位点的磷酸化与GAG起始木糖基转移酶-1（XT-1）和GAG延伸软骨素4-磺基转移酶-1（C4ST-1）的表达之间的关系。和软骨素合成酶1（CHSY-1）基因。凝血酶以相似的时间模式刺激了所有四个靶残基（Thr220，Ser245，Ser250和Ser255残基）的磷酸化-磷酸化在15分钟（研究的最早时间点）达到最大，随后磷蛋白水平下降。以下4小时。Jnk，p38和PI3K，选择性地介导Thr220残基的磷酸化，而丝氨酸残基被多种激酶不同地磷酸化。凝血酶刺激了所有三个基因的表达-XT-1，C4ST-1和CHSY-1。介导Thr220磷酸化的三个途径也参与了XT-1的表达。目标途径（不包括Jnk）参与了GAG延伸基因（C4ST-1和CHSY-1）的表达。这些发现支持以下论点：单个Smad接头区域的磷酸化位点与蛋白聚糖上GAG链的起始和延伸的基因表达有关。这项工作的背景是，GAG延长的特异性抑制剂代表了一种潜在的预防GAG延长和脂质结合的治疗剂，结果表明该途径的特异性使得特异性靶向GAG延长而不干扰其在治疗上是可行的。与蛋白聚糖所涉及的其他生理过程有关。介导Thr220磷酸化的三个途径也参与了XT-1的表达。目标途径（不包括Jnk）参与了GAG延伸基因（C4ST-1和CHSY-1）的表达。这些发现支持以下论点：单个Smad接头区域的磷酸化位点与蛋白聚糖上GAG链的起始和延伸的基因表达有关。这项工作的背景是，GAG延长的特异性抑制剂代表了一种潜在的预防GAG延长和脂质结合的治疗剂，结果表明该途径的特异性使得在不干扰GAG延长的情况下可能具有治疗可行性。与蛋白聚糖所涉及的其他生理过程有关。介导Thr220磷酸化的三个途径也参与了XT-1的表达。目标途径（不包括Jnk）参与了GAG延伸基因（C4ST-1和CHSY-1）的表达。这些发现支持以下论点：单个Smad接头区域的磷酸化位点与蛋白聚糖上GAG链的起始和延伸的基因表达有关。这项工作的背景是，GAG延长的特异性抑制剂代表了一种潜在的预防GAG延长和脂质结合的治疗剂，结果表明该途径的特异性使得特异性靶向GAG延长而不干扰其在治疗上是可行的。与蛋白聚糖所涉及的其他生理过程有关。