osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification

Abstract

Osteoprotegerin (OPG) is a secreted protein that inhibits osteoclast formation. In this study the physiological role of OPG is investigated by generating OPG-deficient mice. Adolescent and adult OPG-/- mice exhibit a decrease in total bone density characterized by severe trabecular and cortical bone porosity, marked thinning of the parietal bones of the skull, and a high incidence of fractures. These findings demonstrate that OPG is a critical regulator of postnatal bone mass. Unexpectedly, OPG-deficient mice also exhibit medial calcification of the aorta and renal arteries, suggesting that regulation of OPG, its signaling pathway, or its ligand(s) may play a role in the long observed association between osteoporosis and vascular calcification.

Figure 1

Gene targeting at the OPG locus. (A) Schematic representation of the genomic structure of the murine OPG locus. Restriction sites are indicated as follows: (RI) EcoRI; (X) XmnI; (P) PstI. Exons are indicated as black boxes and numbered by Roman numerals. Introns are shown as thin black lines. (B) The targeting vector used to disrupt the OPG gene. The PGK–neo casette was placed in reverse orientation within exon 2, replacing the portion of exon 2 encoding the first 93 amino acids of the mature OPG protein. This strategy effectively eliminates the coding region for the first two cysteine-rich domains of OPG that are required for its activity and places a translation stop codon in-frame, preventing translation of any downstream OPG sequence. (C) The structure of a targeted OPG allele following recombination of the targeting vector at the OPG locus. (D) Southern blot analysis of PstI-digested genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) OPG knockout mice. The wild-type allele is a 2.3-kb PstI fragment, and the targeted allele is a 3.0-kb PstI fragment. The small open box in A and C represents the DNA probe used to screen for recombinant ES cell clones. (E) Northern blot analysis of RNA from offspring derived from heterozygous matings. Ten micrograms of total RNA collected from liver was probed for OPG and β-actin expression. The absence of OPG expression in OPG^−/− mice confirmed that OPG is a null allele.

Figure 2

Radiographic analysis of the bones of OPG^−/− and wild-type mice. (A,B) Radiographs of 2-month-old female wild-type and OPG^−/− mice, respectively. (C,D) Radiographs of the leg, hemipelvis and vertebrae of 2-month-old female wild-type and OPG^−/− mice respectively. OPG^−/− mice were x-rayed adjacent to wild-type and heterozygous mice using the same x-ray film, to allow for direct comparison of bone density. The strongest phenotype is evident in the vertebrae and long bones. The cortical bone in the femur and pelvis are thinned, and the femoral growth plate is not visible. OPG^+/− mice are not different from OPG^+/+ mice at this time point. (E) Gross anomalies of the skeleton are seen as early as 1 month after birth in the form of multiple fractures (arrows). (F) Radiograph of 6-month-old female wild-type mouse. (G) Radiograph of 5-month-old OPG^−/− female mouse. Note severe deformity of the vertebral column due to the collapse of several vertebral bodies (arrow). This was confirmed by subsequent lateral radiographs of the spine.

Figure 3

Histological evaluation of bone morphology in 7-day and 2-month-old female OPG^−/− vs. OPG^+/+ mice. (A) OPG^+/+ femur; normal femur morphology. (B) OPG^−/− femur; note minimal loss of the primary and secondary spongiosa. (C) OPG^+/+ femur, cortical area; normal morphology. (D) OPG^−/− femur, cortical area, note somewhat increased bone resorption and remodeling. (E) OPG^+/+ vertebral body; normal morphology. (F) OPG^−/− vertebral body; note osteoporosis characterized by loss of trabecular bone and thinning of the cortical bone. Magnification, 4× in A and B; 30× in C and D; 20× in E and F; H & E stained. (G) OPG^+/+ femur; normal femur morphology. (H) OPG^−/− femur; note marked loss of the primary and secondary spongiosa, and collapse of the normally rounded articular surface. (I) OPG^+/+ femur, cortical area; normal morphology. (J) OPG^−/− femur, cortical area; note severe cortical bone porosity with bone resorption and active remodeling. (K) OPG^+/+ vertebral body; normal morphology. (L) OPG^−/− vertebral body; note severe osteoporosis characterized by loss of trabecular bone and thinning of the cortical bone. Magnification, 4× in G and H; 15× in I and J; 10× in K and L; H & E stained.

Figure 4

Bone remodeling rate, collagen structure, and trabecular density changes in OPG^−/− mice. (A,B) TRAP-stained sections (60×) of the metaphyseal region of the proximal humerus showing increased numbers of osteoclasts (arrows) in the OPG^−/− (B) compared to OPG^+/+ mice (A). (C,D) Polarized light microscopy of cortical bone of the humeral diaphysis showing lamellar collagen deposition in bone of the OPG^+/+ (C) compared to the woven pattern in OPG^−/− mice (D). (E–G) Von Kossa-stained frozen sections of the proximal tibial metaphysis of 6-month-old OPG^+/+, OPG^+/−, and OPG^−/− mice. Note normal morphology in the OPG^+/+ mice (E), lower density in the metaphysis of the OPG^+/− mice (F), and even further reduced density in the OPG^−/− mice (G). Osteoclast surface as a percent of bone surface (OcS%BS) (H) and osteoblast surface as a percent of bone surface (ObS%BS) (I) are increased in the vertebrae, tibial metaphysis (Tibia MP) and the tibial diaphysis (Tibia DP) of the OPG^−/− (solid bars) vs. the OPG^+/+ (shaded bars) mice. (*) Different from OPG^+/+, P < 0.005. (J) Osteocyte number per mm² bone area (OtN/BAr) in the tibial diaphysis is increased in OPG^−/− vs. the OPG^+/+ mice, P < 0.05. (K) Quantitative representation of the trabecular bone density in the metaphyseal region of the tibia expressed as a percent of the total tissue area (BV%TV) in 6-month-old OPG^+/+, OPG^+/−, and OPG^−/− mice. The trabecular bone density was markedly reduced in the proximal tibial metaphysis in the homozygous knockout mice, n = 6, vs. heterozygous mice, n = 9, and wild type mice, n = 7. (*) Significantly lower than wild type, P < 0.001.

Figure 5

Arterial pathology in OPG^−/− mice. (A) In situ hybridization with radioactively labeled antisense OPG probe shows high expression of OPG in the incipient part of the aorta of a E18.5 mouse embryo (arrow). Very high signal is detected in a tangential section of the aorta through the smooth muscle wall (arrowhead). (B) OPG expression is also detected by in situ hybridization in the smooth muscle wall of the renal artery of the adult rat. (C) Note normal morphology of a OPG^+/+ mouse abdominal aorta; H & E stained, magnification, 4×. (D) Normal morphology of the renal artery of an adult OPG^+/+ mouse; H&E stained, 10×. (E) Extensive calcification (arrowhead) in the medial and subintimal region of the ascending aorta of a 2-month-old OPG^−/− mouse. Acute dissection of the aortic wall is marked by an arrow; on the opposite wall of the aorta, granulation tissue is present at the site of a former dissection; H & E stained, 2×. (F) Medial/subintimal calcification (arrow) in the renal artery of a 2-month-old OPG^−/− mouse with moderate intimal proliferation; H & E stained, 20×.