Glycosaminoglycans modulate RANKL-induced osteoclastogenesis

Abstract

Skeletal integrity is tightly regulated by the activity of osteoblasts and osteoclasts that are both under the control of extracellular glycosaminoglycans (GAGs) through their interactions with endogenous growth factors and differentiation-promoting ligands. Receptor activator of NF-kappa-B ligand (RANKL), which is a tumor necrosis factor (TNF)-related protein that is critical for osteoclast formation, is produced by osteoblasts and further modulated by certain types of GAGs. Using unfractionated osteoblast-derived GAGs that reflect the complex tissue microenvironment within which osteoclasts reside, we demonstrate that these GAGs block the osteoclastogenic activity of RANKL. Furthermore, RANKL significantly reduces extracellular signal-regulated protein kinase (ERK) activity, a putative suppressor of osteoclastogenesis, but osteoblast-derived GAGs eliminate the inhibitory effects of RANKL on ERK activity. Notably, while imposing an anti-osteoclastic effect, these GAGs also enhanced the proliferation of osteoblasts. Thus, the osteoblast microenvironment is a potent source of GAGs that promote bone anabolic activities. The anti-osteoclastogenic and osteoblast-related mitogenic activities of these GAGs together may provide a key starting point for the development of selective sugar-based therapeutic compounds for the treatment of osteopenic disorders.

Figure 1

Anion exchange chromatography of soluble and cell surface GAGs from primary osteoblasts. The crude GAGs were purified from porcine primary osteoblast culture media (soluble fraction) or cell extracts (cell surface fraction). A, Samples were applied to a 50 ml CaptoQ-Sepharose column at 4 ml/min in the presence of 1 M NaCl. During washing, a small amount of GAG eluted at absorbance of 232 nm and generated a peak from fraction number 640 to 760. B, Sepharose CL-6B size-exclusion column chromatogram of the GAGs isolated from soluble and cell surface fractions. Size fractions of soluble GAG chains displayed two peaks (1 and 2). Cell surface GAG chains displayed only one peak (2). The deflections below baseline represent monitor artifacts.

Figure 2

Binding ability of GAGs to RANKL. A, Different amounts of RANKL (10 ng, 50 ng and 250 ng) were added to the plate coated with 5 μg soluble or cell surface GAG. An enzyme based assay was performed to detect the amount of RANKL bound to the immobilized GAG. Data represents the mean ± S.E. of triplicate experiments. B, RANKL (10 ng) was added to heparin-Sepharose beads with or without 5 μg/ml free heparin or GAG. Lane 1 was RANKL protein as a positive control. RANKL bound to the beads was visualized by immunoblot analysis and the amount of RANKL protein was determined by densitometric analysis. Data represent the average of three independent experiments.

Figure 3

The effect of GAGs on RANKL-induced osteoclast formation. RAW264.7 cells were cultured for 5 days in the presence of 10 ng/ml RANKL, or 10 ng/ml RANKL together with either 5 μg/ml soluble GAG or 5 μg/ml surface GAG. The cells without any treatment served as the control. Osteoclastogenesis was monitored by TRAP. A, RAW264.7 monocytic cells were negative in TRAP staining (control). After 5 days of treatment with 10 ng/ml RANKL, numerous TRAP positive (purple), multinucleated cells were observed in all cultures with RANKL. All images are from randomly chosen fields using a 10× objective. The data are representative of three independent experiments. B, GAGs from osteoblasts inhibited RANKL-induced osteoclastic differentiation of RAW264.7 cells. Data represent the mean ± S.E. of triplicate experiments.

Figure 4

The effect of soluble GAGs on RANKL-induced ERK activity. RAW264.7 cells were cultured for 5 days in the presence of 10 ng/ml RANKL, or 10 ng/ml RANKL together with 5 μg/ml soluble GAG. Immunoblot analysis reveals that RANKL antagonizes ERK phosphorylation, whereas the osteoblast-derived soluble GAG reduces the inhibitory effect of RANKL on ERK activity. The blot is representative of three independent biological replicates.

Figure 5

The effect of GAGs on osteoblast proliferation. A, Primary human osteoblasts were grown for 2 days with or without 5 μg/ml soluble or cell surface GAG. Cells left untreated served as control. The actin cytoskeleton and the nuclei of the cells were stained with rhodamine phalloidin or DAPI, respectively and the images obtained by confocal microscopy. B, Primary human osteoblasts were deprived of serum to synchronize and arrest the cell growth for 24 hr before treatment with 0.5, 5, 50 μg/ml soluble or cell surface GAGs. After 24 hr, cell proliferation was determined by measuring BrdU incorporation. The cells without any treatment served as the control. Data represent the mean ± S.E. of triplicate experiments.