Transforming growth factor ␤-inducible early gene 1 (TIEG1) is a member of the Krüppel-like transcription factor family. To understand the physiological role of TIEG1, we generated TIEG ؊/؊ (null) mice and found that the TIEG ؊/؊ mice had increased osteoblast numbers with no increased bone formation parameters. However, when calvarial osteoblasts (OBs) were isolated from neonatal TIEG ؊/؊ and TIEG ؉/؉ mice and cultured in vitro, the TIEG ؊/؊ cells displayed reduced expression of important OB differentiation markers. When the OBs were differentiated in vitro by treatment with bone morphogenic protein 2, the OBs from TIEG ؉/؉ calvaria displayed several mineralized nodules in culture, whereas those from TIEG ؊/؊ mice showed no nodules. To characterize the OBs' ability to support osteoclast differentiation, the OBs from TIEG ؉/؉ and TIEG ؊/؊ mice were cultured with marrow and spleen cells from TIEG ؉/؉ mice. Significantly fewer osteoclasts developed when TIEG ؊/؊ OBs were used to support osteoclast differentiation than when TIEG ؉/؉ OBs were used. Examination of gene expression in the TIEG ؊/؊ OBs revealed decreased RANKL and increased OPG expression compared to TIEG ؉/؉ OBs. The addition of RANKL to these cocultures only partially restored the ability of TIEG ؊/؊ OBs to support osteoclast differentiation, whereas M-CSF alone or combined with RANKL had no additional effect on osteoclast differentiation. We conclude from these data that TIEG1 expression in OBs is critical for both osteoblast-mediated mineralization and osteoblast support of osteoclast differentiation.Krüppel-like transcription factors (KLFs) are DNA-binding transcriptional regulators which contain C 2 , H 2 -type zinc fingers and play important roles in regulating biological processes such as cell growth, differentiation, and embryogenesis (1, 5, 32). The number of members of the KLF family has been increasing, and it is estimated that 1% of the human genome might contain this family of regulatory factors (5, 11). Our laboratory has cloned a member of this family, the transforming growth factor ␤ (TGF-␤)-inducible early gene 1 (TIEG1), since it represented a primary response gene to TGF-␤ treatment in human osteoblasts (28). Cook et al. (4) identified TIEG2, which shares 91% homology with TIEG1 within the zinc finger region but only 44% homology at the N terminus region. They also showed evidence that overexpression of TIEG2 in Chinese hamster ovary cells inhibits cell proliferation. Recently, Wang et al. (36) identified another member of the TIEG family, TIEG3, which has properties similar to those of TIEG1 and TIEG2.A better understanding of the mechanism of action of TIEG1 is evolving. Using a GAL4-based transcriptional assay, Cook et al. (4) demonstrated that TIEG1 protein has three repression domains. Studies by Zhang et al. (39) identified an alpha-helical repression motif located within the repression domain of TIEG1 and TIEG2. These authors have also shown evidence that these motifs mediate the direct interaction of TIEG1 with mSin3A, whic...

To better understand the complex roles of transforming growth factor-beta (TGF-β) in bone metabolism, we examined the impact of a range of TGF-β concentrations on osteoclast differentiation. In co-cultures of support cells and spleen or marrow osteoclast precursors, low TGF-β concentrations stimulated while high concentrations inhibited differentiation. We investigated the influences of TGF-β on macrophage colony stimulating factor (M-CSF), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG) expression and found a dose dependent inhibition of M-CSF expression. RANKL expression was elevated at low TGF-β concentrations with a less dramatic increase in OPG. Addition of OPG blocked differentiation at the stimulatory TGF-β dose. Thus, low TGF-β concentrations elevated the RANKL/OPG ratio while high concentrations did not, supporting that, at low TGF-β concentrations, there is sufficient M-CSF and a high RANKL/OPG ratio to stimulate differentiation. At high TGF-β concentrations, the RANKL/OPG ratio and M-CSF expression were both repressed and there was no differentiation. We examined whether TGF-β-mediated repression of osteoclasts differentiation is due to these changes by adding M-CSF and/or RANKL and did not observe any impact on differentiation repression. We studied direct TGF-βimpacts on osteoclast precursors by culturing spleen or marrow cells with M-CSF and RANKL. TGF-β treatment dose-dependently stimulated osteoclast differentiation. These data indicate that low TGF-β levels stimulate osteoclast differentiation by impacting the RANKL/OPG ratio while high TGF-β levels repress osteoclast differentiation by multiple avenues including mechanisms independent of the RANKL/OPG ratio or M-CSF expression regulation.Transforming growth factor-beta (TGF-β) is a ubiquitous multifunctional cytokine that has a spectrum of influences. The variety of reported responses to TGF-β depends, at least in part, on experimental conditions as well as the cell type under study. Within the bone environment, TGF-β is a key regulator of bone metabolism. Although all TGF-β isoforms bind to the same receptor complex, there have been some reports of different cellular responses to the different isoforms (Jennings et al

2-Methoxyestradiol (2-ME), a naturally occurring metabolite of 17beta-estradiol, is highly cytotoxic to a wide range of tumor cells but is harmless to most normal cells. However, 2-ME prevented bone loss in ovariectomized rats, suggesting it inhibits bone resorption. These studies were performed to determine the direct effects of 2-ME on cultured osteoclasts. 2-ME (2 microM) reduced osteoclast number by more than 95% and induced apoptosis in three cultured osteoclast model systems (RAW 264.7 cells cultured with RANKL, marrow cells co-cultured with stromal support cells, and spleen cells cultured without support cells in media supplemented with RANKL and macrophage colony stimulating factor (M-CSF)). The 2-ME-mediated effect was ligand specific; 2-hydroxyestradiol (2-OHE), the immediate precursor to 2-ME, exhibited less cytotoxicity; and 2-methoxyestrone (2-MEOE1) the estrone analog of 2-ME, was not cytotoxic. Co-treatment with ICI 182,780 did not antagonize 2-ME, suggesting that the cytotoxicity was not estrogen receptor-dependent. 2-ME-induced cell death in RAW 264.7 cells coincided with an increase in gene expression of cytokines implicated in inhibition of differentiation and induction of apoptosis. In addition, the 2-ME-mediated decrease in cell survival was partially inhibited by anti-lymphotoxin(LT)beta antibodies, suggesting that 2-ME-dependent effects involve LTbeta. These results suggest that 2-ME could be useful for treating skeletal diseases in which bone resorption is increased, such as postmenopausal osteoporosis and cancer metastasis to bone.

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