Department of Pharmacology, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, P,O,Box 1057 Blindern, N-0316 Oslo, Norway.
J Exp Clin Cancer Res. 2012 Sep 11;31(1):72. doi: 10.1186/1756-9966-31-72.
It is important to understand the mechanisms by which the cells integrate signals from different receptors. Several lines of evidence implicate epidermal growth factor (EGF) receptor (EGFR) in the pathophysiology of hepatocarcinomas. Data also suggest a role of prostaglandins in some of these tumours, through their receptors of the G protein-coupled receptor (GPCR) family. In this study we have investigated mechanisms of interaction between signalling from prostaglandin receptors and EGFR in hepatocarcinoma cells.
The rat hepatocarcinoma cell line MH1C1 and normal rat hepatocytes in primary culture were stimulated with EGF or prostaglandin E2 (PGE2) and in some experiments also PGF2α. DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA, phosphorylation of proteins in signalling pathways was assessed by Western blotting, mRNA expression of prostaglandin receptors was determined using qRT-PCR, accumulation of inositol phosphates was measured by incorporation of radiolabelled inositol, and cAMP was determined by radioimmunoassay.
In the MH1C1 hepatocarcinoma cells, stimulation with PGE2 or PGF2α caused phosphorylation of the EGFR, Akt, and ERK, which could be blocked by the EGFR tyrosine kinase inhibitor gefitinib. This did not occur in primary hepatocytes. qRT-PCR revealed expression of EP1, EP4, and FP receptor mRNA in MH1C1 cells. PGE2 stimulated accumulation of inositol phosphates but not cAMP in these cells, suggesting signalling via PLCβ. While pretreatment with EP1 and EP4 receptor antagonists did not inhibit the effect of PGE2, pretreatment with an FP receptor antagonist blocked the phosphorylation of EGFR, Akt and ERK. Further studies suggested that the PGE2-induced signal was mediated via Ca2+ release and not PKC activation, and that it proceeded through Src and shedding of membrane-bound EGFR ligand precursors by proteinases of the ADAM family.
The results indicate that in MH1C1 cells, unlike normal hepatocytes, PGE2 activates the MEK/ERK and PI3K/Akt pathways by transactivation of the EGFR, thus diversifying the GPCR-mediated signal. The data also suggest that the underlying mechanisms in these cells involve FP receptors, PLCβ, Ca2+, Src, and proteinase-mediated release of membrane-associated EGFR ligand(s).
了解细胞如何整合来自不同受体的信号非常重要。有几条证据表明表皮生长因子受体（EGFR）参与肝癌的病理生理学。数据还表明，前列腺素通过其 G 蛋白偶联受体（GPCR）家族的受体在其中一些肿瘤中发挥作用。在这项研究中，我们研究了前列腺素受体信号与肝癌细胞中 EGFR 信号相互作用的机制。
用 EGF 或前列腺素 E2（PGE2）刺激大鼠肝癌细胞系 MH1C1 和原代培养的正常大鼠肝细胞，在某些实验中也用 PGF2α 刺激。通过掺入放射性标记的胸苷测定 DNA 合成，通过 Western 印迹评估信号通路中蛋白质的磷酸化，使用 qRT-PCR 测定前列腺素受体的 mRNA 表达，通过掺入放射性标记的肌醇测量肌醇磷酸盐的积累，通过放射免疫测定法测定 cAMP。
在 MH1C1 肝癌细胞中，PGE2 或 PGF2α 的刺激导致 EGFR、Akt 和 ERK 的磷酸化，这可以被 EGFR 酪氨酸激酶抑制剂吉非替尼阻断。在原代肝细胞中不会发生这种情况。qRT-PCR 显示 MH1C1 细胞中表达 EP1、EP4 和 FP 受体 mRNA。PGE2 刺激这些细胞中肌醇磷酸盐的积累，但不刺激 cAMP 的积累，表明通过 PLCβ 进行信号转导。虽然 EP1 和 EP4 受体拮抗剂的预处理不能抑制 PGE2 的作用，但 FP 受体拮抗剂的预处理阻断了 EGFR、Akt 和 ERK 的磷酸化。进一步的研究表明，PGE2 诱导的信号是通过 Ca2+释放介导的，而不是通过 PKC 激活介导的，并且它通过 Src 和 ADAM 家族的蛋白水解酶介导膜结合的 EGFR 配体前体的脱落进行。
结果表明，在 MH1C1 细胞中，与正常肝细胞不同，PGE2 通过 EGFR 的转激活激活 MEK/ERK 和 PI3K/Akt 途径，从而使 GPCR 介导的信号多样化。数据还表明，这些细胞中的潜在机制涉及 FP 受体、PLCβ、Ca2+、Src 和蛋白水解酶介导的膜相关 EGFR 配体（s）的释放。
