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

Abstract

Id proteins play important roles in osteogenic differentiation; however, the molecular mechanism remains unknown. In this study, we established that inhibitor of differentiation (Id) proteins, including Id1, Id2, and Id3, associate with core binding factor ␣-1 (Cbfa1) to cause diminished transcription of the alkaline phosphatase (ALP) and osteocalcin (OCL) gene, leading to less ALP activity and osteocalcin (OCL) production. Id acts by inhibiting the sequence-specific binding of Cbfa1 to DNA and by decreasing the expression of Cbfa1 in cells undergoing osteogenic differentiation. p204, an interferon-inducible protein that interacts with both Cbfa1 and Id2, overcame the Id2-mediated inhibition of Cbfa1-induced ALP activity and OCL production. We show that 1) p204 disturbed the binding of Id2 to Cbfa1 and enabled Cbfa1 to bind to the promoters of its target genes and 2) that p204 promoted the translocation from nucleus to the cytoplasm and accelerated the degradation of Id2 by ubiquitin-proteasome pathway during osteogenesis. Nucleus export signal (NES) of p204 is required for the p204-enhanced cytoplasmic translocation and degradation of Id2, because a p204 mutant lacking NES lost these activities. Together, Cbfa1, p204, and Id proteins form a regulatory circuit and act in concert to regulate osteoblast differentiation. INTRODUCTIONThe differentiation of uncommitted mesenchymal cells to osteoblasts is a fundamental process in embryonic development and bone repair. The bone morphogenetic proteins (BMPs) are important regulators of this process (Heldin et al., 1997;Miyazono et al., 2000). They function by binding cell surface receptors; signaling by Smad proteins; and activating bone-specific genes, including core binding factor ␣-1 (Cbfa1) (Ducy, 2000;Miyazono et al., 2001;Attisano and Wrana, 2002). This process is positively or negatively regulated by a variety of coactivators and corepressors (Ducy et al., 2000;Miyazono et al., 2001;Attisano and Wrana, 2002). Cbfa1, also known as Runx2, PeBP2␣A, Osf2, or AML3, is a member of the runt family of transcription factors. It is an essential transcription factor of osteoblast and bone formation (Ducy, 2000). During skeletal development, Cbfa1 is observed first in early mesenchymal condensations, and it is then principally expressed in osteoblasts . Cbfa1 Ϫ/Ϫ mice exhibit a complete lack of ossification, and they die immediately after birth (Komori et al., 1997). Cbfa1 maintains osteoblastic function by regulating the expression of several bone-specific genes such as osteopontin and osteocalcin and by controlling bone extracellular matrix deposition (Ducy, 2000). Mutations in Cbfa1 are found in 65-80% of individuals with cleidocranial dysplasia (CCD) (Lee et al., 1997;Mundlos and Olsen, 1997;Zhou et al., 1999). The molecular mechanisms underlying the pathogenesis of CCD are not completely defined. 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 Sma...

XBP1S Associates with RUNX2 and Regulates Chondrocyte Hypertrophy

“…The medium was replaced approximately every 2-3 days. To test the effect of overexpression of XBP1S protein on chondrogenesis, C3H10T1/ 2 cells were infected with XBP1S expression adenovirus (16,17) or control GFP adenovirus before micromass culture. To test the effect of knocking down XBP1S on chondrogenesis, C3H10T1/2 cells were infected with Ad-XBP1S siRNA or control RFP adenovirus before micromass culture.…”

Cbfa1-dependent expression of an interferon-inducible p204 protein is required for chondrocyte differentiation

“…We previously reported that p204 associates with Cbfa1 and plays an important role in osteoblast differentiation. 12,24,25 Given that Cbfa1 is also required for the hypertrophic chondrocyte differentiation, we sought to determine whether p204 was also involved in the chondrocyte differentiation. We first examined the expression of p204 in growth plate chondrocytes using immunohistochemistry on tibial growth plates of mouse embryos on postcoital day 18.5.…”

“…p204, which interacts with both Cbfa1 and Id2, overcame the Id2-mediated inhibition of Cbfa1-mediated osteogenesis. 25 It remains to be determined whether Ids and pRb are expressed in the hypertrophic chondrocytes and participate in the regulation of chondrocyte hypertrophy by Cbfa1 and p204.…”

“…The media was replaced approximately every 2-3 days. To test the effect of overexpression of p204 protein on chondrogenesis, C3H10T1/2 cells were infected with p204 expression adenovirus 24,25 or control GFP adenovirus at the multiplicity of infection of 50 before micromass culture. To test the effect of knocking down p204 on chondrogenesis, C3H10T1/2 cells were transfected with p204 siRNA expression plasmid (pSuper-p204) or control plasmid (pSuper) before micromass culture.…”

“…23 We previously reported that p204 acted as a transcriptional coactivator of Cbfa1 and therefore enhanced osteoblast differentiation 12 and that pRb is an essential linker between p204 and Cbfa1 thereby increasing its activity; 24 p204, pRb, Cbfa1 and Ids form a protein interaction network and act in concert in regulating the differentiation of pluripotent C2C12 to osteoblasts. 25 In this study, we describe the expression and function of p204 in hypertrophic chondrocyte differentiation as well as the molecular mechanism involved.…”