A CCAAT/enhancer binding protein beta isoform, liver-enriched inhibitory protein, regulates commitment of osteoblasts and adipocytes

Abstract

Although both osteoblasts and adipocytes have a common origin, i.e., mesenchymal cells, the molecular mechanisms that define the direction of two different lineages are presently unknown. In this study, we investigated the role of a transcription factor, CCAAT/enhancer binding protein beta (C/EBPbeta), and its isoform in the regulation of balance between osteoblast and adipocyte differentiation. We found that C/EBPbeta, which is induced along with osteoblast differentiation, promotes the differentiation of mesenchymal cells into an osteoblast lineage in cooperation with Runx2, an essential transcription factor for osteogenesis. Surprisingly, an isoform of C/EBPbeta, liver-enriched inhibitory protein (LIP), which lacks the transcriptional activation domain, stimulates transcriptional activity and the osteogenic action of Runx2, although LIP inhibits adipogenesis in a dominant-negative fashion. Furthermore, LIP physically associates with Runx2 and binds to the C/EBP binding element present in the osteocalcin gene promoter. These data indicate that LIP functions as a coactivator for Runx2 and preferentially promotes the osteoblast differentiation of mesenchymal cells. Thus, identification of a novel role of the C/EBPbeta isoform provides insight into the molecular basis of the regulation of osteoblast and adipocyte commitment.

FIG. 1.

C/EBPβ expression associated with osteoblastogenesis. Expression of C/EBPβ and its isoform, LIP, in mouse primary osteoblasts as determined by immunoblotting analysis (A) and immunochytochemical analysis (B) is shown. C/EBPβ expression (C) and ALP activity (D) of C3H10T1/2 or primary mesenchymal cells cultured with or without BMP2 for 7 days are also shown.

FIG. 2.

C/EBPβ promotes osteoblast differentiation of mesenchymal cells. (A through C) C3H10T1/2 cells were infected with control or C/EBPβ adenovirus at an MOI of 40 and incubated with BMP2 for 7 days. The cells were determined by immunoblotting (A), ALP staining (B), or ALP activity (C). (D) C3H10T1/2 cells were infected with control, Runx2, or C/EBPβ adenovirus at an MOI of 40 and cultured for 7 days. Osteocalcin, type IA collagen (Col IA), or ALP expression in the cells was assessed by RT-PCR. (E) ST2, C2C12, and mouse primary mesenchymal cells were infected with control or C/EBPβ adenovirus at an MOI of 40 and incubated for 7 days. The cells were subjected to ALP staining.

FIG. 3.

C/EBPβ regulates osteoblastogenesis in a Runx2-dependent and -independent fashion. (A) C3H10T1/2 cells were infected with control or C/EBPβ adenovirus with or without Runx2 adenovirus and cultured for 7 days. ALP activity in the cells was measured. (B) Osteocalcin gene promoter fused to the luciferase reporter construct and TK-renilla reporter constructs were transfected into C3H10T1/2 cells together with pcDNA3 (control), the C/EBPβ expression vector, the Runx2 expression vector, or both vectors. Luciferase activity of the cell lysates was measured in relative light units (RLU). (C) The lysates of C3H10T1/2 cells infected with control or C/EBPβ adenovirus together with or without Runx2 adenovirus were examined by immunoblotting with anti-Runx2 or C/EBPβ antibody. (D) The lysates of C3H10T1/2 cells infected with control or C/EBPβ adenovirus were incubated with a biotinylated probe containing the C/EBP binding element (BE) in osteocalcin gene promoter in the presence or absence of unbiotinylated probe (Comp). Associated protein with biotinylated probe was determined by immunoblotting with anti-C/EBPβ antibody. Ppt, precipitation. (E) Luciferase reporter construct-fused osteocalcin gene promoter containing osteocalcin luciferase (OC-Luc) or lacking the C/EBP binding element [OC(ΔC/EBP-BE)-Luc] and the TK-renilla reporter construct were transfected into C3H10T1/2 cells together with pcDNA3 (control), the C/EBPβ expression vector, the Runx2 expression vector, or both vectors. Luciferase activity of the cell lysates was measured. (F) C3H10T1/2 cells were infected with the control, C/EBPβ adenovirus, Runx2 adenovirus, or both adenovirus strains. The cell lysates were immunoprecipitated (IP) with anti-C/EBPβ antibody, and immunoprecipitates were determined by immunoblotting with anti-Runx2 antibody. (G) C3T10T1/2 cells were infected with adenoviruses as indicated in the text and cultured for 7 days. ALP activity in the cells was measured. (H) Mesenchymal cells isolated from Runx2-deficient mice were infected with adenoviruses as indicated in the text and cultured for 7 days. ALP activity of the cells was measured.

FIG. 4.

LIP inhibits adipocyte differentiation in a dominant-negative fashion. (A) Schematic structure of C/EBPβ, LIP, and its deletion mutants. AD, activation domain; BD, binding domain; LZ, leucine zipper. (B) C3H10T1/2 cells infected with control, C/EBPβ, or LIP adenovirus were analyzed by immunoblotting with anti-C/EBPβ antibody. (C) C3H10T1/2 cells were infected with adenoviruses as indicated in Materials and Methods, incubated for 7 days, and then stained with Oil red O. Oil red O-stained area was measured as described in Materials and Methods. (D) PPARγ gene promoter fused to the luciferase reporter construct and TK-renilla reporter constructs was transfected into C3H10T1/2 cells together with expression vectors as indicated. Luciferase activity of the cell lysates was measured. (E) C3H10T1/2 cells were incubated with BMP2 as indicated, and expression of LIP and C/EBPβ was determined by immunoblotting. (F) C3H10T1/2 cells infected with control or Flag-tagged LIP adenovirus were incubated with BMP2 for 7 days and stained with Oil red O. The Oil red O-stained area was measured. (G and H) C3H10T1/2 cells infected with control or Flag-tagged LIP adenovirus were incubated with BMP2 for 7 days and ALP activity (G) and osteocalcin production (H) were determined. (I) C3H10T1/2 cells infected with control or Flag-tagged LIP adenovirus were incubated with BMP2 for 7 days, and the cell lysates were examined by immunoblotting with anti-Flag, Runx2, or C/EBPβ antibody. (J) C2C12 cells infected with control or Flag-tagged LIP adenovirus were incubated with BMP2 for 7 days, and ALP activity was determined.

FIG. 5.

LIP enhances osteogenic action of Runx2 through its physical association. (A) Total RNA of C3H10T1/2 cells infected with control (Cont) or Flag-LIP adenovirus or incubated with BMP2 was determined by RT-PCR analysis. (B) C3H10T1/2 cells were infected with adenoviruses as indicated in the text and incubated for 7 days. ALP activity of the cells was measured. (C) Osteocalcin gene promoters fused to luciferase reporter construct and TK-renilla reporter constructs were transfected into C3H10T1/2 cells together with pcDNA3 (control), LIP expression vector, Runx2 expression vector, or both vectors. Luciferase activity of the cell lysates was measured. (D) C2C12 cells infected with adenovirus as indicated in the text were cultured for 7 days and then examined by ALP staining. (E) C3H10T1/2 cells were infected with control, LIP, or Runx2 adenovirus or both LIP and Runx2 adenovirus. The cell lysates were immunoprecipitated with anti-C/EBPβ antibody, and immunoprecipitates (IP) were determined by immunoblotting with anti-Runx2 antibody. (F) The lysates of Cos7 cells were transfected with Myc-tagged LIP constructs as indicated and incubated with Flag-tagged Runx2 protein immobilized on protein G-agarose beads. Precipitated proteins (Ppt) were examined by immunoblotting with anti-Myc antibody. (G) The lysates of Cos7 cells were transfected with Flag-tagged Runx2 constructs as indicated and incubated with Myc-tagged LIP protein immobilized on protein G-agarose beads. Precipitated proteins were examined by immunoblotting with anti-Flag antibody. (H) The lysates of C3H10T1/2 cells infected with control or LIP adenovirus were incubated with biotinylated probe containing C/EBP binding element (BE) in the osteocalcin gene. Associated protein with biotinylated probe was determined by immunoblotting with anti-C/EBPβ antibody. (I) The lysates of C3H10T1/2 cells infected with control or Flag-tagged LIP adenovirus were subjected to chromatin immunoprecipitation analysis. C, immunoprecipitation with control mouse IgG; F, immunoprecipitation with anti-Flag antibody. (J) C3H10T1/2 cells were infected with adenoviruses as indicated in the text. Seven days after culture, ALP activity was examined. (K) Runx2-deficient mesenchymal cells were infected with control, either LIP adenovirus or Runx2 adenovirus, or both adenoviruses and then cultured for 7 days. ALP activity was determined.