Endogenous Production of TGF-β Is Essential for Osteoclastogenesis Induced by a Combination of Receptor Activator of NF-κB Ligand and Macrophage-Colony- Stimulating Factor

Abstract

Differentiation of osteoclasts, the cells primarily responsible for bone resorption, is controlled by a variety of osteotropic hormones and cytokines. Of these factors, receptor activator of NF-κB (RANK) ligand (RANKL) has been recently cloned as an essential inducer of osteoclastogenesis in the presence of M-CSF. Here, we isolated a stroma-free population of monocyte/macrophage (M/Mφ)-like hemopoietic cells from mouse unfractionated bone cells that were capable of differentiating into mature osteoclasts by treatment with soluble RANKL (sRANKL) and M-CSF. However, the efficiency of osteoclast formation was low, suggesting the requirement for additional factors. The isolated M/Mφ-like hemopoietic cells expressed TGF-β and type I and II receptors of TGF-β. Therefore, we examined the effect of TGF-β on osteoclastogenesis. TGF-β with a combination of sRANKL and M-CSF promoted the differentiation of nearly all M/Mφ-like hemopoietic cells into cells of the osteoclast lineage. Neutralizing anti-TGF-β Ab abrogated the osteoclast generation. These TGF-β effects were also observed in cultures of unfractionated bone cells, and anti-TGF-β blocked the stimulatory effect of 1,25-dihydroxyvitamin D3. Translocation of NF-κB into nuclei induced by sRANKL in TGF-β-pretreated M/Mφ-like hemopoietic cells was greater than that in untreated cells, whereas TGF-β did not up-regulate the expression of RANK, the receptor of RANKL. Our findings suggest that TGF-β is an essential autocrine factor for osteoclastogenesis.

Transforming Growth Factor-β Induces Expression of Receptor Activator of NF-κB Ligand in Vascular Endothelial Cells Derived from Bone

“…Thus, in pathological conditions, especially osteolytic bone metastasis, vascular endothelial cells might work dominantly and accelerate bone resorption in vivo. Moreover, TGF-␤ promoted the growth of BDECs (data not shown), which is consistent with the previous reports that it induced angiogenesis (40,41); it would lead to further osteolysis, following tumor growth. TGF-␤ is also known to induce parathyroid hormone-related protein production in breast cancer cells, which in turn upregulates RANKL expression in osteoblasts (16).…”

Low-density Lipoprotein Receptor Deficiency Causes Impaired Osteoclastogenesis and Increased Bone Mass in Mice because of Defect in Osteoclastic Cell-Cell Fusion

“…After culturing the cells for 3 days, the mean maximum diameter and area of LDLR Ϫ/Ϫ multinucleated osteoclasts decreased by 40 and 60%, respectively, when compared with osteoclasts derived from wild-type littermates. Furthermore, the fusion index, which was expressed as the mean number of nuclei per multinucleated osteoclast (55), in cultures of LDLR Ϫ/Ϫ osteoclast precursors was ϳ20% of that observed in cultures of wild-type osteoclast precursors (Fig. 4D).…”

Identification and Characterization of the Precursors Committed to Osteoclasts Induced by TNF-Related Activation-Induced Cytokine/Receptor Activator of NF-κB Ligand

“…To obtain BMMs, mouse bone marrow cells were collected from the tibiae and femora of the mice, and cultured for 3 days in ␣-MEM containing 10% FBS and M-CSF (50 ng/ml) in 100-mm-diameter type I collagen-coated culture dishes (IWAKI-Asahi Glass) (1 ϫ 10 7 cells/10 ml/dish). To promote the efficiency of osteoclast differentiation, human TGF-␤ (1 ng/ml) was added to the culture medium together with M-CSF, according to the previous reports (25)(26)(27)(37)(38)(39). After culturing for 3 days, floating cells were gently removed by rinsing with PBS, and cells remaining attached to the culture plates were collected by treatment with trypsin-EDTA (Invitrogen Life Technologies) and used as BMMs.…”