Parturition Research Group, Department of Surgery and Cancer, Imperial College London, London, England.
PLoS One. 2012;7(11):e50734. doi: 10.1371/journal.pone.0050734. Epub 2012 Nov 30.
15-deoxy-Δ 12,14- Prostaglandin J2 (15dPGJ2) inhibits Nuclear factor kappa B (NF-κB) in human myocytes and amniocytes and delays inflammation induced preterm labour in the mouse. 15dPGJ2 is a ligand for the Chemoattractant Receptor Homologous to the T helper 2 cell (CRTH2), a G protein-coupled receptor, present on a subset of T helper 2 (Th2) cells, eosinophils and basophils. It is the second receptor for Prostaglandin D2, whose activation leads to chemotaxis and the production of Th2-type interleukins. The cellular distribution of CRTH2 in non-immune cells has not been extensively researched, and its identification at the protein level has been limited by the lack of specific antibodies. In this study we explored the possibility that CRTH2 plays a role in 15dPGJ2-mediated inhibition of NF-κB and would therefore represent a novel small molecule therapeutic target for the prevention of inflammation induced preterm labour.
The effect of a small molecule CRTH2 agonist on NF-κB activity in human cultured amniocytes and myocytes was assessed by detection of p65 and phospho-p65 by immunoblot. Endogenous CRTH2 expression in amniocytes, myocytes and peripheral blood mononuclear cells (PBMCs) was examined by PCR, western analysis and flow cytometry, with amniocytes and myocytes transfected with CRTH2 acting as a positive control in flow cytometry studies.
The CRTH2 agonist had no effect on NF-κB activity in amniocytes and myocytes. Although CRTH2 mRNA was detected in amniocytes and myocytes, CRTH2 was not detectable at the protein level, as demonstrated by western analysis and flow cytometry. 15dPGJ2 inhibited phospho-65 in PBMC'S, however the CRTH2 antagonist was not able to attenuate this effect. In conclusion, CRTH2 is not expressed on human amniocytes or myocytes and plays no role in the mechanism of 15dPGJ2-mediated inhibition of NF-κB.
15-脱氧-Δ12,14-前列腺素 J2（15dPGJ2）可抑制人肌细胞和成纤维细胞中的核因子 κB（NF-κB），并延缓小鼠的炎症性早产。15dPGJ2 是趋化因子受体同源物 2（CRTH2）的配体，CRTH2 是一种 G 蛋白偶联受体，存在于部分辅助性 T 细胞 2（Th2）细胞、嗜酸性粒细胞和嗜碱性粒细胞上。它是前列腺素 D2 的第二种受体，其激活可导致趋化和产生 Th2 型白细胞介素。CRTH2 在非免疫细胞中的细胞分布尚未得到广泛研究，并且由于缺乏特异性抗体，其在蛋白质水平上的鉴定受到限制。在这项研究中，我们探讨了 CRTH2 在 15dPGJ2 介导的 NF-κB 抑制中的作用的可能性，因此它可能成为预防炎症性早产的新型小分子治疗靶标。
通过免疫印迹检测 p65 和磷酸化 p65 来评估小分子 CRTH2 激动剂对人培养的成纤维细胞和肌细胞中 NF-κB 活性的影响。通过 PCR、western 分析和流式细胞术检测成纤维细胞、肌细胞和外周血单核细胞（PBMC）中的内源性 CRTH2 表达，并用成纤维细胞和肌细胞转染 CRTH2 作为流式细胞术研究中的阳性对照。
CRTH2 激动剂对成纤维细胞和肌细胞中的 NF-κB 活性没有影响。尽管在成纤维细胞和肌细胞中检测到了 CRTH2 mRNA，但通过 western 分析和流式细胞术检测到 CRTH2 蛋白水平无法检测到。15dPGJ2 抑制了 PBMCs 中的磷酸化 65，但 CRTH2 拮抗剂不能减弱这种作用。总之，CRTH2 不表达于人成纤维细胞或肌细胞上，并且在 15dPGJ2 介导的 NF-κB 抑制机制中不起作用。
