成骨细胞衍生的转化生长因子-β1通过激活蛋白-1（AP-1）和核因子-κB（NF-κB）刺激人癌细胞释放白细胞介素-8（IL-8）。

Osteoblast-derived TGF-beta1 stimulates IL-8 release through AP-1 and NF-kappaB in human cancer cells.

作者信息

Fong Yi-Chin, Maa Ming-Chei, Tsai Fuu-Jen, Chen Wen-Chi, Lin Jaung-Geng, Jeng Long-Bin, Yang Rong-Sen, Fu Wen-Mei, Tang Chih-Hsin

机构信息

Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan.

出版信息

J Bone Miner Res. 2008 Jun;23(6):961-70. doi: 10.1359/jbmr.080206.

DOI:10.1359/jbmr.080206

PMID:18435575

Abstract

INTRODUCTION

The bone marrow microenvironment is further enriched by growth factors released during osteoclastic bone resorption. It has been reported that the chemokine interleukin (IL)-8 is a potent and direct activator of osteoclastic differentiation and bone resorption. However, the effect of bone-derived growth factors on the IL-8 production in human cancer cells and the promotion of osteoclastogenesis are largely unknown. The aim of this study was to investigate whether osteoblast-derived TGF-beta1 is associated with osteolytic bone diseases.

MATERIALS AND METHODS

IL-8 mRNA levels were measured using RT-PCR analysis. MAPK phosphorylation was examined using the Western blot method. siRNA was used to inhibit the expression of TGF-beta1, BMP-2, and IGF-1. DNA affinity protein-binding assay and chromatin immunoprecipitation assays were used to study in vitro and in vivo binding of c-fos, c-jun, p65, and p50 to the IL-8 promoter. A transient transfection protocol was used to examine IL-8, NF-kappaB, and activator protein (AP)-1 activity.

RESULTS

Osteoblast conditioned medium (OBCM) induced activation of IL-8, AP-1, and NF-kappaB promoter in human cancer cells. Osteoblasts were transfected with TGF-beta1, BMP-2, or IGF-1 small interfering RNA, and the medium was collected after 48 h. TGF-beta1 but not BMP-2 or IGF-1 siRNA inhibited OBCM-induced IL-8 release in human cancer cells. In addition, TGF-beta1 also directly induced IL-8 release in human cancer cells. Activation of AP-1 and NF-kappaB DNA-protein binding and MAPKs after TGF-beta1 treatment was shown, and TGF-beta1-induced IL-8 promoter activity was inhibited by the specific inhibitors of MAPK cascades.

CONCLUSIONS

In this study, we provide evidence to show that the osteoblasts release growth factors, including TGF-beta1, BMP-2, and IGF-1. TGF-beta1 is the major contributor to the activation of extracellular signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), leading to the activation of AP-1 and NF-kappaB on the IL-8 promoter and initiation of IL-8 mRNA and protein release, thereby promoting osteoclastogenesis.

摘要

引言

破骨细胞性骨吸收过程中释放的生长因子可进一步丰富骨髓微环境。据报道，趋化因子白细胞介素（IL）-8是破骨细胞分化和骨吸收的强效直接激活剂。然而，骨源性生长因子对人癌细胞中IL-8产生及破骨细胞生成促进作用的影响在很大程度上尚不清楚。本研究的目的是调查成骨细胞衍生的转化生长因子-β1（TGF-β1）是否与溶骨性骨疾病相关。

材料与方法

采用逆转录聚合酶链反应（RT-PCR）分析检测IL-8 mRNA水平。使用蛋白质免疫印迹法检测丝裂原活化蛋白激酶（MAPK）磷酸化情况。使用小干扰RNA（siRNA）抑制TGF-β1、骨形态发生蛋白-2（BMP-2）和胰岛素样生长因子-1（IGF-1）的表达。采用DNA亲和蛋白结合试验和染色质免疫沉淀试验研究c-fos、c-jun、p65和p50在体外和体内与IL-8启动子的结合情况。采用瞬时转染方案检测IL-8、核因子κB（NF-κB）和激活蛋白（AP）-1活性。

结果

成骨细胞条件培养基（OBCM）可诱导人癌细胞中IL-8、AP-1和NF-κB启动子的激活。用TGF-β1、BMP-2或IGF-1小干扰RNA转染成骨细胞，48小时后收集培养基。TGF-β1小干扰RNA而非BMP-2或IGF-1小干扰RNA可抑制OBCM诱导的人癌细胞中IL-8释放。此外，TGF-β1还可直接诱导人癌细胞中IL-8释放。结果显示TGF-β1处理后AP-1和NF-κB DNA-蛋白结合及MAPKs激活，且TGF-β1诱导的IL-8启动子活性被MAPK级联反应的特异性抑制剂抑制。

结论

在本研究中，我们提供证据表明成骨细胞释放包括TGF-β1、BMP-2和IGF-1在内的生长因子。TGF-β1是细胞外信号调节激酶（ERK）、p38和c-Jun氨基末端激酶（JNK）激活的主要促成因素，导致IL-8启动子上AP-1和NF-κB激活以及IL-8 mRNA和蛋白释放启动，从而促进破骨细胞生成。