Protease activated receptors (PARs) transactivate both epidermal growth factor receptors (EGFR) and transforming growth factor (TGF)-β receptors (TGFBR1) in vascular smooth muscle leading to the increased expression of genes (CHST11 and CHSY1) which are rate limiting for the enzymes that mediate hyperelongation of glycosaminoglycan (GAG) chains on the lipid-binding proteoglycan, biglycan. This is an excellent model to investigate mechanisms of transactivation as the processes are biochemically distinct. EGFR transactivation is dependent on the classical matrix metalloprotease (MMP) based triple membrane bypass mechanism and TGFBR1 transactivation is dependent on Rho/ROCK signalling and integrins. We have shown that all kinase receptor signalling is targeted towards phosphorylation of the linker region of the transcription factor, Smad2. We investigated the mechanisms of thrombin mediated kinase receptor transactivation signalling using anti-phospho antibodies and Western blotting and gene expression by RT-PCR. Thrombin stimulation of phospho-Smad2 (Ser 245/250/255) and of phospho-Smad2(Thr220) via EGFR transactivation commences quickly and extends out to at least 4 h whereas transactivation via TGFBR1 is delayed for 120 min but also persists for at least 4 h. Signalling of thrombin stimulated Smad linker region phosphorylation is approximately equally inhibited by the MMP inhibitor, GM6001 and the ROCK inhibitor, Y27632, and similarly expression of CHST11 and CHSY1 is approximately equally inhibited by GM6001 and Y27632. The data establishes Smad linker region phosphorylation as a central target of all transactivation signalling of GAG gene expression and thus an upstream kinase may be a target to prevent all transactivation signalling and its pathophysiological consequences.

中文翻译：

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PAR-1 介导的激酶受体反式激活机制：Smad 接头区域磷酸化。

蛋白酶激活受体 (PAR) 反式激活血管平滑肌中的表皮生长因子受体 (EGFR) 和转化生长因子 (TGF)-β 受体 (TGFBR1)，导致基因 (CHST11 和 CHSY1) 的表达增加，这些基因对介导脂质结合蛋白聚糖（双糖链蛋白聚糖）上的糖胺聚糖 (GAG) 链超伸长的酶。这是一个很好的模型来研究反式激活机制，因为这些过程在生化上是不同的。EGFR 反式激活依赖于基于经典基质金属蛋白酶 (MMP) 的三膜旁路机制，而 TGFBR1 反式激活依赖于 Rho/ROCK 信号传导和整合素。我们已经表明，所有激酶受体信号传导都针对转录因子 Smad2 的接头区域的磷酸化。我们使用抗磷酸化抗体和蛋白质印迹法研究了凝血酶介导的激酶受体反式激活信号传导的机制，并通过 RT-PCR 进行基因表达。通过 EGFR 反式激活对磷酸化 Smad2 (Ser 245/250/255) 和磷酸化 Smad2 (Thr220) 的凝血酶刺激开始迅速并延长至至少 4 小时，而通过 TGFBR1 的反式激活延迟 120 分钟但也持续至少4小时。MMP 抑制剂 GM6001 和 ROCK 抑制剂 Y27632 几乎同样地抑制了凝血酶刺激的 Smad 接头区域磷酸化的信号，并且类似地，GM6001 和 Y27632 也同样地抑制了 CHST11 和 CHSY1 的表达。