Osteoblast-Derived TGF-β1 Stimulates IL-8 Release Through AP-1 and NF-κB in Human Cancer Cells

Abstract

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. 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. 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. 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.