TIEG1 Null Mouse-Derived Osteoblasts Are Defective in Mineralization and in Support of Osteoclast Differentiation In Vitro

Abstract

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...

Kruppel-like Factors (KLFs) in muscle biology

“…This transcription factor was first described in osteoblasts as a "TGF-β Inducible Early Gene" and thus carried the name (TIEG-1) [55]. Subsequent studies from the laboratory or Dr. Thomas Spelsberg have revealed an important role for this factor in the regulation of bone mineralization [56], osteoclast differentiation [56] and epithelial proliferation [57,58]. KLF10 can modulate Smad signaling in osteoblasts and mice with systemic KLF10 deficiency are osteopenic [59] and have impaired tendon healing in response to mechanical injury [60].…”

TIEG-null mice display an osteopenic gender-specific phenotype

“…TIEG -/-mice were originally developed in a C57BL/6:129SVJ mixed breed background as described previously [22]. To derive TIEG -/-mice in a congenic background, we bred our mixed breed TIEG -/-animals against C57BL/6 WT mice for at least 10 generations.…”

“…Calvarial OBs, isolated from the TIEG -/-mice revealed decreased expression of important OB marker genes, including osteocalcin, osterix, and alkaline phosphatase [22]. Coculture studies with osteoclast (OC) precursor cells demonstrated that fewer mature OCs developed when TIEG -/-OBs were used to support OC differentiation compared to that of wild-type (WT) OBs [22]. Gene expression analysis of TIEG -/-OBs revealed a decrease in the expression of RANKL and an increase in the expression of OPG relative to WT OBs, which could well explain the decreased support of OC differentiation by TIEG -/-OBs [22].…”

“…Since TGFβ is known to play important roles in multiple facets of bone biology, and since we have shown that TIEG is an important regulator of TGFβ signaling in OBs, and can mimic TGFβ action when overexpressed, we developed a TIEG knockout mouse model (TIEG -/-) [22]. These animals were originally developed in a C57BL/6:129SVJ mixed breed genetic background.…”

“…These animals were originally developed in a C57BL/6:129SVJ mixed breed genetic background. Calvarial OBs, isolated from the TIEG -/-mice revealed decreased expression of important OB marker genes, including osteocalcin, osterix, and alkaline phosphatase [22]. Coculture studies with osteoclast (OC) precursor cells demonstrated that fewer mature OCs developed when TIEG -/-OBs were used to support OC differentiation compared to that of wild-type (WT) OBs [22].…”