EGF-like Ligands Stimulate Osteoclastogenesis by Regulating Expression of Osteoclast Regulatory Factors by Osteoblasts: IMPLICATIONS FOR OSTEOLYTIC BONE METASTASES

Ji Zhu,Xun Jia,Guozhi Xiao,Yibin Kang,Nicola C Partridge,Ling Qin

doi:10.1074/jbc.m705064200

Abstract

Epidermal growth factor (EGF)-like ligands and their receptors constitute one of the most important signaling networks functioning in normal tissue development and cancer biology. Recent in vivo mouse models suggest this signaling network plays an essential role in bone metabolism. Using a coculture system containing bone marrow macrophage and osteoblastic cells, here we report that EGF-like ligands stimulate osteoclastogenesis by acting on osteoblastic cells. This stimulation is not a direct effect because osteoclasts do not express functional EGF receptors (EGFRs). Further studies reveal that EGF-like ligands strongly regulate the expression of two secreted osteoclast regulatory factors in osteoblasts by decreasing osteoprotegerin (OPG) expression and increasing monocyte chemoattractant protein 1 (MCP1) expression in an EGFR-dependent manner and consequently stimulate TRAP-positive osteoclast formation. Addition of exogenous OPG completely inhibited osteoclast formation stimulated by EGF-like ligands, while addition of a neutralizing antibody against MCP-1 exhibited partial inhibition. Coculture with bone metastatic breast cancer MDA-MB-231 cells had similar effects on the expression of OPG and MCP1 in the osteoblastic cells, and those effects could be partially abolished by the EGFR inhibitor PD153035. Because a high percentage of human carcinomas express EGF-like ligands, our findings suggest a novel mechanism for osteolytic lesions caused by cancer cells metastasizing to bone.

Highlights

The adult human skeleton continuously undergoes remodeling, namely, being resorbed by osteoclasts and renewed by osteoblasts
The EGFlike ligands consist of epidermal growth factor (EGF), amphiregulin, and transforming growth factor ␣ (TGF␣), which only bind to the EGF receptors (EGFRs), and heparin-binding EGF (HB-EGF), betacellulin, and epiregulin, which can bind to both the EGFR and ErbB4
We demonstrate that EGF-like ligands have the ability to strongly stimulate osteoclastogenesis by regulating OPG and monocyte chemoattractant protein 1 (MCP1) expression in osteoblastic cells in an EGFR-dependent manner

Summary

EXPERIMENTAL PROCEDURES

Chemicals—Recombinant human EGF, TGF␣, HB-EGF, amphiregulin, heregulin were purchased from R&D Systems (Minneapolis, MN). To obtain primary osteoclastic cultures, bone marrow cells were flushed out from femora and tibiae of a 1–2-monthold mouse, plated in coculture medium (␣MEM supplemented with 10% heat-inactivated fetal bovine serum, 100 international units/ml penicillin, 100 ␮g/ml streptomycin, and 2 mM L-glutamine) in a 100-mm dish, and incubated at 37 °C in 5% CO2 overnight. The day, the nonadherent cells were pelleted and seeded at a density of 200,000 cells/cm2 These cells are considered as BMM, the osteoclast precursors, and cultured in the presence of sRANKL (30 ng/ml) and M-CSF (30 ng/ml) for 5 days with a medium change at day 3 to obtain mature osteoclasts. For coculture experiments of MC3T3 cells and osteoclasts, MC3T3 cells were seeded on day 0 in coculture medium plus 50 ␮g/ml L-ascorbic acid. Statistical analyses were carried out using the Student’s t test (Microsoft Excel 2002)

RESULTS

When cells were incubated with

Regulatory Factors in Osteoblastic

DISCUSSION