School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC 3083, Australia.
School of Pharmacy, The University of Queensland, Wooloongabba, QLD 4102, Australia.
Cell Signal. 2018 Jul;47:37-43. doi: 10.1016/j.cellsig.2018.03.009. Epub 2018 Mar 22.
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.
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。许多生长因子参与了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通路尚未得到很好的定义。本研究调查了凝血酶介导的角质形成细胞信号转导。凝血酶受体，蛋白酶激活受体 1（PAR-1）是一种七跨膜 G 蛋白偶联受体，已知可转激活表皮生长因子受体（EGFR）。永生化人角质形成细胞（HaCaT 细胞）用凝血酶和 EGFR 和 MAP 激酶的选择性抑制剂处理。用 SDS-PAGE 分离全细胞裂解物，并使用针对转录因子 Smad2 的抗体通过 Western blot 进行分析。使用实时定量聚合酶链反应测量 PAI-1 的 mRNA 表达，并用划痕伤口测定测量角质形成细胞迁移。Western blot 数据显示，凝血酶介导 PAR-1 对 EGFR 的转激活以及转录因子 Smad2 连接子（Smad2L）区域的下游磷酸化。UO126 抑制 ERK1/2 导致 Smad2L 磷酸化减少，而 p38 抑制剂 SB202190 和 JNK 抑制剂 SP600125 则没有。这种凝血酶介导的途径特异性地使 Smad2L Ser250 磷酸化，而 Ser245 和 Ser255 则没有。凝血酶增加 PAI-1 mRNA 表达和角质形成细胞迁移，而当阻断 EGFR 或 ERK1/2 时则减少。综上所述，这些结果表明凝血酶介导的 PAI-1 在角质形成细胞中的 mRNA 表达和迁移是通过 EGFR 转激活发生的，涉及信号转导中间物 ERK1/2 和 Smad2，并且可能是皮肤伤口愈合的关键途径。
