Cianfrocca Roberta, Tocci Piera, Semprucci Elisa, Spinella Francesca, Di Castro Valeriana, Bagnato Anna, Rosanò Laura
Laboratory of Molecular Pathology, Regina Elena National Cancer Institute, Rome, Italy.
Laboratory of Molecular Pathology, Regina Elena National Cancer Institute, Rome, Italy.
Life Sci. 2014 Nov 24;118(2):179-84. doi: 10.1016/j.lfs.2014.01.078. Epub 2014 Feb 12.
In epithelial ovarian cancer (EOC), activation of endothelin-1 (ET-1)/endothelin A receptor (ETAR) signalling is linked to many tumor promoting effects, such as proliferation, angiogenesis, invasion and metastasis. These effects are dependent by the activation of critical signalling pathways, such as MAPK, Akt, and β-catenin, through specific cytosolic and nuclear scaffolding functions of β-arrestin 1 (β-arr1). Here, we have assessed the potential role of ET-1/ETAR in promoting NF-κB signalling in EOC cells through β-arr-1 recruitment.
We used cultured HEY EOC cells cultured in the presence or absence of ET-1 and the ETAR antagonist BQ123. The phosphorylation of p65 and Iκ-Bα was evaluated by immunoblotting analysis. The interaction between p65 and β-arr1 was evaluated by immunoprecipitation experiments in nuclear extracts. NF-κB promoter activity was evaluated by transfection with NF-κB-driven luciferase reporter construct. Assessment of the function of β-arr1 was achieved by β-arr1 silencing with shRNA and expression of β-arr1-FLAG expression vector.
In EOC cells, ET-1 promotes the phosphorylation of p65 subunit and the cytoplasmic inhibitor IκB that in turn led to increased NF-κB transcriptional activity. These effects were inhibited by the use of BQ123, as well as by β-arr-1 silencing, suggesting that ET-1 through ETAR promotes the recruitment of β-arr1 to regulate NF-κB signalling. Moreover, the nuclear physical interaction between p65 and β-arr1 indicates a nuclear function of β-arr-1 in ETAR-driven NF-κB transcriptional activity.
Altogether these findings reveal a previously unrecognized pathway that depends on β-arr1 to sustain NF-κB signalling in response to ETAR activation in ovarian cancer.
在上皮性卵巢癌（EOC）中，内皮素-1（ET-1）/内皮素A受体（ETAR）信号通路的激活与多种肿瘤促进作用相关，如增殖、血管生成、侵袭和转移。这些作用依赖于关键信号通路的激活，如MAPK、Akt和β-连环蛋白，通过β-抑制蛋白1（β-arr1）的特定胞质和核支架功能实现。在此，我们评估了ET-1/ETAR通过β-arr-1募集在促进EOC细胞中NF-κB信号传导方面的潜在作用。
我们使用在有或无ET-1及ETAR拮抗剂BQ123存在的情况下培养的HEY EOC细胞。通过免疫印迹分析评估p65和Iκ-Bα的磷酸化。通过核提取物中的免疫沉淀实验评估p65与β-arr1之间的相互作用。通过用NF-κB驱动的荧光素酶报告构建体转染来评估NF-κB启动子活性。通过用shRNA沉默β-arr1和表达β-arr1-FLAG表达载体来评估β-arr1的功能。
在EOC细胞中，ET-1促进p65亚基和细胞质抑制剂IκB的磷酸化，进而导致NF-κB转录活性增加。使用BQ123以及β-arr-1沉默可抑制这些作用，表明ET-1通过ETAR促进β-arr1的募集以调节NF-κB信号传导。此外，p65与β-arr1之间的核物理相互作用表明β-arr-1在ETAR驱动的NF-κB转录活性中具有核功能。
这些发现共同揭示了一条先前未被认识的途径，该途径依赖于β-arr1来维持卵巢癌中对ETAR激活的NF-κB信号传导。
