CCAAT/Enhancer-binding protein homologous protein (CHOP) decreases bone formation and causes osteopenia

Abstract

CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP), is a member of the C/EBP family of nuclear proteins and plays a role in osteoblastic and adipocytic cell differentiation. CHOP is necessary for normal bone formation, but the consequences of its overexpression in vivo are not known. To investigate the direct actions of CHOP on bone remodeling in vivo, we generated transgenic mice overexpressing CHOP under the control of the human osteocalcin promoter. CHOP transgenics exhibited normal weight and reduced bone mineral density. Static and dynamic femoral bone histomorphometry revealed that CHOP overexpression caused reduced trabecular bone volume, secondary to decreased bone formation rates. One of 2 lines displayed a decrease in the number of osteoblasts, but in vivo bromodeoxyuridine labeling demonstrated that CHOP overexpression did not have an effect on osteoblastic cell replication. The decreased osteoblast cell number was accounted by an increase in apoptosis, as determined by DNA fragmentation measured by transferase-mediated digoxigenin-deoxyuridine triphosphate (dUTP) in situ nick-end labeling (TUNEL) reaction. In conclusion, transgenic mice overexpressing CHOP in the bone microenvironment have impaired osteoblastic function leading to osteopenia.

Figure 1

Schematic representation of the construct used for the creation of transgenic mice, and relative levels of CHOP transgene and endogenous mRNA expression in CHOP homozygous (Line 1) and heterozygous (Line 2) transgenic mice and wild type (WT) controls. Total RNA was extracted from calvariae of 4 week old CHOP transgenics and controls, resolved by Northern blot analysis and hybridized with [α-³²P]-labeled CHOP and mouse 18S cDNAs.

Figure 2

Bone mineral density (BMD gm × cm² × 10⁻³), weight (gm) and total body fat (%) of 4 week-old CHOP transgenic heterozygous (Line 2; black bars) and wild type control (WT, white bars) male mice. Values represent means ± SEM; n = 6–7. ^*Significantly different from wild type controls, p<0.05.

Figure 3

Static bone histomorphometry performed on femurs from 4 week old male CHOP transgenic heterozygous (Line 2; black bars) and wild type controls (WT, white bars). Stained sections were examined, and trabecular bone volume (BV/TV, %), osteoblast and osteoclast number per perimeter (Ob/B.Pm./mm; Oc/B.Pm./mm) trabecular number (Tb.N.;/mm) and thickness (Tb.Th.; μm) and eroded surface/bone surface (ES/BS) determined. Values are means + SEM; n = 6–7. *Significantly different from WT controls, p<0.05. The lower panel shows representative femoral sections from CHOP transgenics and WT controls stained with Von Kossa and examined at a final magnification of 40x.

Figure 4

Dynamic bone histomorphometry performed on femurs from 4 week old male CHOP heterozygous transgenic (Line 2; black bars) and wild type controls (WT, white bars). Unstained sections were analyzed by fluorescence microscopy and mineralizing surface per bone surface (MS/BS; %), mineral apposition rate (MAR; μm/day) and bone formation rate/bone surface (BFR/BS; μm³/μm²/day) determined. Values are means ± SEM; n = 6–7. *Significantly different from WT controls, p<0.05. The lower panels show representative femoral sections from CHOP transgenics and WT controls and visualization of calcein and demeclocycline labels by fluorescence microscopy at a final magnification of 100x and 400x.

Figure 5

Effect of CHOP overexpression on osteoblast cell proliferation and apoptosis in femoral sections from 4 week-old male CHOP heterozygous transgenics (Line 2) and wild type (WT) controls. A. For visualization of BrdU incorporation, mice were injected intraperitoneally with BrdU, 50 μg/gram of body weight, 24 h before sacrifice and processed as described in Materials and Methods. Arrows point to the BrdU labeled cells in the primary spongiosa at a magnification of 400x. B. DNA fragmentation was detected by the TUNEL reaction, and osteoblasts, identified as cells lining the trabecular surface, in which the nuclei were dark brown rather than blue-green were considered apoptotic. Final magnification 200x. Data are means ± SEM; n = 3–4, and are expressed as the percent apoptotic/total cells from CHOP transgenics (black bars) and a WT control (white bars). *Significantly different from WT controls, p<0.05.