CCAAT/enhancer-binding protein homologous protein (CHOP) regulates osteoblast differentiation

Abstract

Differentiation of committed osteoblasts is controlled by complex activities involving signal transduction and gene expression, and Runx2 and Osterix function as master regulators for this process. Recently, CCAAT/enhancer-binding proteins (C/EBPs) have been reported to regulate osteogenesis in addition to adipogenesis. However, the roles of C/EBP transcription factors in the control of osteoblast differentiation have yet to be fully elucidated. Here we show that C/EBP homologous protein (CHOP; also known as C/EBPzeta) is expressed in bone as well as in mesenchymal progenitors and primary osteoblasts. Overexpression of CHOP reduces alkaline phosphatase activity in primary osteoblasts and suppresses the formation of calcified bone nodules. CHOP-deficient osteoblasts differentiate more strongly than their wild-type counterparts, suggesting that endogenous CHOP plays an important role in the inhibition of osteoblast differentiation. Furthermore, endogenous CHOP induces differentiation of calvarial osteoblasts upon bone morphogenetic protein (BMP) treatment. CHOP forms heterodimers with C/EBPbeta and inhibits the DNA-binding activity as well as Runx2-binding activity of C/EBPbeta, leading to inhibition of osteocalcin gene transcription. These findings indicate that CHOP acts as a dominant-negative inhibitor of C/EBPbeta and prevents osteoblast differentiation but promotes BMP signaling in a cell-type-dependent manner. Thus, endogenous CHOP may have dual roles in regulating osteoblast differentiation and bone formation.

FIG. 1.

Profiles of expression of C/EBP family members in neonatal tissues and mesenchymal progenitor cells. (A) RT-PCR performed on cDNA derived from various neonatal mouse tissues. RT-PCR experiments were performed using primers specific for CHOP, C/EBPα, C/EBPβ, C/EBPδ, and HPRT1. Amplification of CHOP, C/EBPα, C/EBPβ, or C/EBPδ was performed with 32 cycles. Amplification of a HPRT1 fragment was used to confirm equal levels of target cDNA in the samples. (B) RT-PCR performed on cDNA derived from several mesenchymal cells cultured with or without BMP-2 (300 ng/ml) for 5 days or from 3T3-L1 cells cultured with or without adipogenic cocktail DIII for 2 days. Amplification of CHOP, C/EBPα, C/EBPβ, or C/EBPδ was performed with 30 cycles. MSC, marrow stromal cells. (C) C2C12 cells were cultured with or without BMP-2 (300 ng/ml) for 4 days, and total cell lysates were immunoblotted with anti-GADD153 (anti-CHOP) antibody, anti-C/EBPβ antibody, or antitubulin antibody. Lysate of 3T3-L1 cells which express CHOP and C/EBPβ was loaded as a positive control. The asterisk indicates a nonspecific band.

FIG. 2.

CHOP suppresses adipocyte differentiation. (A) Calvarial osteoblasts were infected with LacZ (MOI = 200), FLAG-CHOP (F-CHOP) (MOI = 200), or FLAG-C/EBPβ (MOI = 100) adenovirus and cultured for 3 days. Then, nuclear extracts from infected cells were immunoblotted with anti-FLAG antibody to determine the expression levels of induced proteins. (B) 3T3-L1 cells were infected with LacZ adenovirus, C/EBPβ adenovirus, or both at an MOI of 100 for each and incubated with or without DIII cocktail for 4 days. Then, cells were examined for adipogenic differentiation by Nile red staining and real-time RT-PCR for aP2 and adipsin. mRNA values were normalized to the amounts of HPRT1. (C) 3T3-L1 cells were infected with LacZ adenovirus, FLAG-C/EBPβ adenovirus, or both at an MOI of 100 for each and incubated for 14 days. Then, expression levels of PPARγ2 and adipsin were assessed by real-time RT-PCR. mRNA values were normalized to the amounts of HPRT1.

FIG. 3.

CHOP inhibits differentiation of calvarial osteoblasts. Primary calvarial osteoblasts were infected with LacZ or CHOP adenovirus at an MOI of 200 for each. (A) Osteoblasts were cultured with or without 50 μg/ml AA plus β-GP for 16 days. ALP staining and ALP activity are shown. p-NP, p-nitrophenylphosphate.(B) Osteoblasts were cultured with or without AA-β-GP and 300 ng/ml BMP-2 for 7 days. (C and D) Quantitative RT-PCR for osteocalcin and Runx2 was performed, and values were normalized to the amounts of HPRT1. a.a., AA; β-gp, β-GP; OC, osteocalcin.

FIG. 4.

Endogenous CHOP has a dual effect on primary osteoblast differentiation. (A) Expression levels of the CHOP protein in wild-type and CHOP-null calvarial osteoblasts were assessed by immunoblot analysis and compared to that in C2C12 cells stimulated by BMP-2. Nuclear extracts were immunoblotted with anti-CHOP antibody and antilamin antibody (for a loading control). (B) Primary calvarial osteoblasts from two groups of littermates (four wild-type and three knockout mice total) were cultured without any stimulants for 6 days. Cells were subjected to ALP staining. (C) Total RNA from parallel samples shown in panel B was subjected to quantitative RT-PCR individually for Runx2, ALP, and osteocalcin, and values were normalized to the amounts of HPRT1. The expression levels of the two genotypes were compared and significance was determined by t testing. (D) Wild-type (litter II #7) and homozygous CHOP-null (litter II #6) osteoblasts were infected with LacZ or CHOP adenovirus at an MOI of 200 and subsequently cultured in normal medium for 11 days. ALP staining is shown. (E) Wild-type (litter II #7) and homozygous CHOP-null (litter II #6) osteoblasts were infected with LacZ or CHOP adenovirus at an MOI of 200 and cultured with AA-β-GP plus 100 ng/ml BMP-2 for 4 days (ALP staining) or 11 days (von Kossa staining). (F) Osteoblasts treated in parallel with those shown in panels D and E were subjected to quantitative RT-PCR for ALP and osteocalcin at day 4. a.a., AA; β-gp, β-GP; OC, osteocalcin.

FIG. 5.

Inhibition of adipogenic differentiation by endogenous CHOP. (A) Primary fibroblasts from three individual wild-type and CHOP knockout littermates were infected or not with CHOP adenovirus and cultured with or without BMP-2 (100 ng/ml) for 10 days. Then, cells were examined for adipogenic differentiation by Nile red staining. (B) Primary fibroblasts from CHOP^−/− and CHOP^+/+ littermates were infected or not with CHOP adenovirus and cultured with or without BMP-2 (100 ng/ml) for 5 days. Then, levels of expression of PPARγ2 and aP2 were assessed by quantitative RT-PCR. mRNA values were normalized to the amounts of HPRT1.

FIG. 6.

CHOP interferes with cooperative induction of the osteocalcin gene by Runx2 and C/EBPβ. (A) C2C12 cells were infected with LacZ or Runx2 adenovirus in combination with LacZ or CHOP virus at an MOI of 100 for each (total, 200 MOI per well) and cultured in normal medium for 7 days and subsequently ALP stained and subjected to quantitative RT-PCR for osteocalcin (upper panels). C2C12 cells were transfected with the osteocalcin gene promoter fused to the luciferase reporter construct and the phRL-TK-renilla reporter together with Runx2 and/or CHOP cDNAs (lower panel). Osteocalcin-Luc activity levels were normalized to the level of phRL-TK-renilla activity. (B) Primary fibroblasts were infected with the indicated adenoviruses at the MOIs shown and then subjected to quantitative RT-PCR for osteocalcin (day 5) and ALP staining (day 7). (C) C3H10T1/2 cells were transfected with the osteocalcin gene luciferase reporter and the phRL-TK-renilla reporter together with combinations of the indicated cDNAs. OC, osteocalcin.

FIG. 7.

CHOP prevents interaction between Runx2 and C/EBPβ as well as the binding of C/EBPβ to the C/EBP-binding element of the osteocalcin promoter. (A) COS7 cells were transfected with various combinations of plasmids for C/EBPβ, Runx2, or CHOP. The cell lysates were immunoprecipitated (IP) with anti-FLAG antibody and subsequently immunoblotted with anti-Myc antibody. (B) The lysates of COS7 cells transfected with pcDNA3 (control), 6× Myc-C/EBPβ, or both 6× Myc-C/EBPβ and Myc-CHOP were incubated with or without a biotinylated probe containing the C/EBP-binding element in the osteocalcin gene promoter (C/EBP-BE^OG2). Proteins bound to the biotinylated probe were determined by immunoblotting with anti-Myc antibody. Ppt, precipitation. (C) Nuclear extracts from primary calvarial osteoblasts infected with indicated adenoviruses were subjected to EMSA with the biotinylated CEBP-BE^OG2 probe. Unlabeled 200 molar excess oligonucleotides (wild-type [Wt] and mutant [Mut]) were added as competitors. Anti-C/EBPβ antibody was used for the supershift experiment. (D) C2C12 cells were infected or not with CHOP adenovirus and cultured with or without BMP-2 (300 ng/ml) for 2 days. The lysates were subjected to ChIP. PCR amplification was performed using the primers specific for the osteocalcin promoter. α-, anti-; IgG, control mouse immunoglobulin G; N.C., negative control (water).

FIG. 8.

Proposed model for the dual function of CHOP in osteoblast differentiation. Runx2 plays indispensable roles in commitment and maturation of osteoblasts. C/EBPβ cooperates with Runx2 to accelerate osteoblast differentiation. CHOP blocks C/EBPβ from accessing Runx2 and binding the promoters of osteogenic marker genes. BMP signaling positively acts on osteoblast differentiation. Under such conditions, CHOP enhances BMP signaling indirectly, resulting in accelerated osteoblast differentiation.