Regulation of osteoclastogenesis and RANK expression by TGF-beta 1

Transforming growth factor-beta (TGF-beta) has been shown to both inhibit a nd to stimulate bone resorption and osteoclastogenesis. This may be due, in part, to differential effects on bone marrow stromal cells that support os teoclastogenesis vs. direct effects on osteoclastic precursor cells. in the present study, we used the murine monocytic cell line, RAW 264.7, to defin e direct effects of TGF-beta on pre-osteoclastic cells. In the presence of macrophage-colony stimulating factor (M-CSF) (20 ng/ml) and receptor activa tor of NF-kappaB ligand (RANK-L) (50 ng/ml), TGF-beta (0.01-5 ng/ ml) dose- dependently stimulated (by up to 120-fold) osteoclast formation (assessed b y the presence of tartrate-resistant acid phosphatase (TRAP) positive multi nucleated cells and expression of calcitonin and vitronectin receptors). In addition, TGF-beta1 also increased steady state RANK mRNA levels in a time - (by up to 3.5-fold at 48 h) and close-dependent manner (by up to 2.2-fold at 10 ng/ml). TGF-beta1 induction of RANK mRNA levels was present both in undifferentiated RAW cells as well as in cells that had been induced to dif ferentiate into osteoclasts by a 7-day treatment with M-CSF and RANK-L. Usi ng a fluorescence-labeled RANK-L probe, we also demonstrated by flow cytome try that TGF-beta1 resulted in a significant increase in the percentage of RANK+RAW cells (P <0.05), as well as an increase in the fluorescence intens ity per cell (P <0.05), the latter consistent with an increase in RANK prot ein expression per cell. These data thus indicate that TGF-beta directly st imulates osteoclastic differentiation, and this is accompanied by increased RANK mRNA and protein expression. (C) 2001 Wiley-Liss, Inc.