The Interferon-inducible p204 Protein Acts as a Transcriptional Coactivator of Cbfa1 and Enhances Osteoblast Differentiation

Abstract

The differentiation of uncommitted mesenchymal cells into osteoblasts is a fundamental molecular event governing both embryonic development and bone repair. The bone morphogenetic proteins (BMPs) are important regulators of this process; they function by binding to cell surface receptors and signaling by means of Smad proteins. Core binding factor ␣-1 (Cbfa1), a member of the runt family of transcription factors, is an essential transcriptional regulator of osteoblast differentiation and bone formation, and this process is positively or negatively regulated by a variety of coactivators and corepressors. We report that p204, an interferon-inducible protein that was previously shown to inhibit cell proliferation and promote the differentiation of myoblasts to myotubes, is a novel regulator in the course of osteogenesis. p204 is expressed in embryonic osteoblasts and hypertrophic chondrocytes in the growth plate as well as in the calvaria osteoblasts of neonatal mice. Its level is increased in the course of the BMP-2-triggered osteoblast differentiation of pluripotent C2C12 cells. This increase is probably due to the activation of the gene encoding 204 (Ifi204) by Smad transcription factor, including Smad1, -4, and -5. Overexpression of p204 enhances the BMP-2-induced osteoblast differentiation in vitro, as revealed by elevated alkaline phosphatase activity and osteocalcin production. p204 acts as a cofactor of Cbfa1: 1) high levels of p204 augment, whereas the lowering of p204 level decreases, the Cbfa1-dependent transcription, and 2) p204 associates with Cbfa1 both in vitro and in vivo. Two nonoverlapping segments in p204 bind to Cbfa1, and the N-terminal 88-amino acid segment of Cbfa1 is required for binding to p204. p204, which is the first interferoninducible protein found to associate with Cbfa1, functions as a novel regulator of osteoblast differentiation.

p204 Protein Overcomes the Inhibition of Core Binding Factor α-1–mediated Osteogenic Differentiation by Id Helix-Loop-Helix Proteins

“…These results, together with the demonstration that Cbfa1 and Id2 form a complex in vivo (Figure 2), indicate that the endogenous Id2 prevents the binding of Cbfa1 to DNA via forming Id2/Cbfa1 complex at early stage of osteogenic differentiation. In accordance with our previously report (Liu et al, 2005), anti-p204 also could precipitate osteocalcin promoter DNA at 48 h time point in that p204 and Cbfa1 form an activation complex in the osteocalcin promoter during osteoblast differentiation. …”

“…We previously reported that p204 acted as a transcriptional coactivator of Cbfa1 and therefore enhanced osteoblast differentiation (Liu et al, 2005) and that pRb is an essential linker between p204 and Cbfa1, thereby increasing its activity (Luan et al, 2007) during the differentiation of pluripotent C2C12 to osteoblast induced by BMP-2. In this study, we report that Id proteins directly associate with Cbfa1 and inhibit Cbfa1-mediated osteogenic differentiation and that p204 overcomes these inhibitions by Id proteins, and we focus on the molecular mechanisms underlying these processes.…”

“…Some mutations of Cbfa1 abolish its DNA binding activity (Lee et al, 1997;Zhou et al, 1999) and others disturb the association of Cbfa1 to its binding partners, including Smads (Xie et al, 2000). It was found that several Cbfa1-binding proteins such as retinoblastoma protein (pRb) (Thomas et al, 2001), p204 (Liu et al, 2005), and Core binding factor ␤ ( Kundu et al, 2002;Yoshida et al, 2002) may also play critical roles in bone development.…”

“…p204 may also play an important role in lymphocytic differentiation based on the observation that p204 mRNA levels are higher in less mature double-positive (CD4 ϩ CD8 ϩ ) thymocytes than in single-positive (CD4 ϩ or CD8ϩ) thymocytes (Deftos et al, 2000). Notch1, an essential factor for promoting the maturation of T thymocytes, can transcriptionally up-regulate p204 gene expression during the maturation to both CD4 ϩ and CD8 ϩ singlepositive lineages of CD4 ϩ CD8 ϩ double-positive thymocytes (Deftos et al, 2000).We previously reported that p204 acted as a transcriptional coactivator of Cbfa1 and therefore enhanced osteoblast differentiation (Liu et al, 2005) and that pRb is an essential linker between p204 and Cbfa1, thereby increasing its activity (Luan et al, 2007) during the differentiation of pluripotent C2C12 to osteoblast induced by BMP-2. In this study, we report that Id proteins directly associate with Cbfa1 and inhibit Cbfa1-mediated osteogenic differentiation and that p204 overcomes these inhibitions by Id proteins, and we focus on the molecular mechanisms underlying these processes.…”

“…Some mutations of Cbfa1 abolish its DNA binding activity (Lee et al, 1997;Zhou et al, 1999) and others disturb the association of Cbfa1 to its binding partners, including Smads (Xie et al, 2000). It was found that several Cbfa1-binding proteins such as retinoblastoma protein (pRb) (Thomas et al, 2001), p204 (Liu et al, 2005), and Core binding factor ␤ ( Kundu et al, 2002;Yoshida et al, 2002) may also play critical roles in bone development.Inhibitor of differentiation (Id) proteins belong to the helix-loop-helix (HLH) family. Id proteins function as global regulators of gene expression during cell growth and differentiation (reviewed in Ruzinova and Benezra, 2003;Wong et al, 2004).…”