TGF-β and RANKL utilize overlapping intracellular signaling cascades [3]. These regulate osteoclastic gene zvadfmk via key transcription factors such as NFATc1 and c-fos. Like TGF-β, these transcription factors are essential for osteoclast formation [12] and [13]. It is possible, therefore, that TGF-β’s enabling and augmentative effect could be mediated via an action on these transcription factors. To investigate this, it was first necessary to determine the time-period during which TGF-β enables osteoclastogenesis, and establish whether TGF-β-induced signals are essential at the inception of osteoclastogenesis or necessary throughout the differentiation process. Once this had been established, the ability of TGF-β to modify NFATc1 and c-fos expression during this period was examined.
Materials and methods
Media and reagents. Stroma depleted nonadherent, M-CSF dependent bone marrow monocytes (BMM) and RAW264.7 murine monocytes (ATCC, UK) were incubated in MEM and Earle’s salts (EMEM) supplemented with 10% FCS (Autogen Bioclear, UK), 2 mmol/l glutamine, 100 IU/ml benzylpenicillin, and 100 mg/ml streptomycin (all from Sigma, UK). Incubations were performed at 37 °C in 5% CO2, and cultures fed every 2–3 days. Recombinant human M-CSF, soluble human recombinant RANKL, recombinant human OPG, and murine recombinant TGF-β1 were obtained from Insight Biotechnology. Anti-TNF-α antibody and pan-specific TGF-β antibody were purchased from R&D systems. Anti-NFATc1 antibody was obtained from Santa Cruz Biotechnology. All other reagents were obtained from Sigma unless stated.
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RNA isolation and characterization Total RNA was
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