Keratinocyte proliferation and migration is essential during re-epithelialisation for the restoration of the epithelial barrier during skin wound healing. Numerous growth factors are involved in the stimulation of keratinocyte proliferation and migration. The signalling pathways that drive these processes during wound healing are not well defined. This study investigated thrombin-mediated signalling in keratinocytes. The thrombin receptor, protease-activated receptor 1 (PAR-1) is a seven transmembrane G-protein coupled receptor that is known to transactivate the epidermal growth factor receptor (EGFR). Immortalized human keratinocytes (HaCaT cells) were treated with thrombin and selective inhibitors to EGFR and MAP kinases. Whole cell lysates were separated on SDS-PAGE and analysed by Western blot using antibodies against transcription factor Smad2. Quantitative real-time polymerase chain reaction was used to measure the mRNA expression of PAI-1 while scratch wound assays were used to measure keratinocyte migration. Western blot data showed that thrombin mediates PAR-1 transactivation of EGFR and the downstream phosphorylation of the transcription factor Smad2 linker (Smad2L) region. ERK1/2 inhibition by UO126 caused a decrease in Smad2L phosphorylation while the p38 inhibitor SB202190 and JNK inhibitor SP600125 did not. Smad2L Ser250 was specifically phosphorylated by this thrombin mediated pathway while Ser245 and Ser255 were not. Thrombin increased PAI-1 mRNA expression and keratinocyte migration and this was reduced when either EGFR or ERK1/2 were blocked. Taken together these results show that thrombin mediated mRNA expression of PAI-1 in keratinocytes and migration occurs via EGFR transactivation and involves signalling intermediates ERK1/2 and Smad2 and may be a key pathway in skin wound healing.

在上皮再形成过程中，角质形成细胞的增殖和迁移对于皮肤伤口愈合过程中上皮屏障的恢复至关重要。许多生长因子参与角质形成细胞增殖和迁移的刺激。在伤口愈合过程中驱动这些过程的信号传导途径尚不明确。这项研究调查了凝血酶介导的角质形成细胞中的信号传导。凝血酶受体，蛋白酶激活受体1（PAR-1）是七种跨膜G蛋白偶联受体，已知可以激活表皮生长因子受体（EGFR）。用凝血酶和EGFR和MAP激酶选择性抑制剂处理永生化的人角质形成细胞（HaCaT细胞）。全细胞裂解物在SDS-PAGE上分离，并使用抗转录因子Smad2的抗体通过Western blot分析。定量实时聚合酶链反应用于测量PAI-1的mRNA表达，而刮擦试验用于测量角质形成细胞迁移。Western印迹数据显示，凝血酶介导EGFR的PAR-1反式激活和转录因子Smad2接头（Smad2L）区的下游磷酸化。UO126抑制ERK1 / 2导致Smad2L磷酸化降低，而p38抑制剂SB202190和JNK抑制剂SP600125却没有。Smad2L Ser250被该凝血酶介导的途径特异性磷酸化，而Ser245和Ser255没有。凝血酶增加PAI-1 mRNA表达和角质形成细胞迁移，而当EGFR或ERK1 / 2被阻断时，凝血酶降低。