Jin Won Jong, Kim Bongjun, Kim Darong, Park Choo Hea-Young, Kim Hong-Hee, Ha Hyunil, Lee Zang Hee
Department of Cell and Developmental Biology, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea.
Division of Life and Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Republic of Korea.
Exp Mol Med. 2017 Feb 17;49(2):e295. doi: 10.1038/emm.2016.148.
The chemokine CXCL10 and its receptor CXCR3 play a role in breast cancer metastasis to bone and osteoclast activation. However, the mechanism of CXCL10/CXCR3-induced intracellular signaling has not been fully investigated. To evaluate CXCL10-induced cellular events in the mouse breast cancer cell line 4T1, we developed a new synthetic CXCR3 antagonist JN-2. In this study, we observed that secretion of CXCL10 in the supernatant of 4T1 cells was gradually increased during cell growth. JN-2 inhibited basal and CXCL10-induced CXCL10 expression and cell motility in 4T1 cells. Treatment of 4T1 cells with CXCL10 increased the expression of P65, a subunit of the NF-κB pathway, via activation of the NF-κB transcriptional activity. Ectopic overexpression of P65 increased CXCL10 secretion and blunted JN-2-induced suppression of CXCL10 secretion, whereas overexpression of IκBα suppressed CXCL10 secretion. These results indicate that the CXCL10/CXCR3 axis creates a positive feedback loop through the canonical NF-κB signaling pathway in 4T1 cells. In addition, treatment of osteoblasts with conditioned medium from JN-2-treated 4T1 cells inhibited the expression of RANKL, a crucial cytokine for osteoclast differentiation, which resulted in an inhibitory effect on osteoclast differentiation in the co-culture system of bone marrow-derived macrophages and osteoblasts. Direct intrafemoral injection of 4T1 cells induced severe bone destruction; however, this effect was suppressed by the CXCR3 antagonist via downregulation of P65 expression in an animal model. Collectively, these results suggest that the CXCL10/CXCR3-mediated NF-κB signaling pathway plays a role in the control of autonomous regulation of CXCL10 and malignant tumor properties in breast cancer 4T1 cells.
趋化因子CXCL10及其受体CXCR3在乳腺癌向骨转移及破骨细胞激活过程中发挥作用。然而，CXCL10/CXCR3诱导的细胞内信号传导机制尚未得到充分研究。为了评估CXCL10在小鼠乳腺癌细胞系4T1中诱导的细胞事件，我们研发了一种新型合成CXCR3拮抗剂JN-2。在本研究中，我们观察到4T1细胞上清液中CXCL10的分泌在细胞生长过程中逐渐增加。JN-2抑制4T1细胞中基础及CXCL10诱导的CXCL10表达和细胞运动。用CXCL10处理4T1细胞通过激活NF-κB转录活性增加了NF-κB途径亚基P65的表达。P65的异位过表达增加了CXCL10的分泌，并减弱了JN-2诱导的CXCL10分泌抑制，而IκBα的过表达则抑制了CXCL10的分泌。这些结果表明，CXCL10/CXCR3轴在4T1细胞中通过经典NF-κB信号通路形成了一个正反馈环。此外，用来自经JN-2处理的4T1细胞的条件培养基处理成骨细胞，抑制了破骨细胞分化关键细胞因子RANKL的表达，这导致在骨髓来源巨噬细胞和成骨细胞共培养系统中对破骨细胞分化产生抑制作用。直接股内注射4T1细胞会诱导严重的骨破坏；然而，在动物模型中，这种作用被CXCR3拮抗剂通过下调P65表达所抑制。总的来说，这些结果表明CXCL10/CXCR3介导的NF-κB信号通路在控制乳腺癌4T1细胞中CXCL10的自主调节和恶性肿瘤特性方面发挥作用。
