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. 2005 Sep;115(9):2402-11.
doi: 10.1172/JCI24918.

Osteoblast-derived PTHrP is a potent endogenous bone anabolic agent that modifies the therapeutic efficacy of administered PTH 1-34

Dengshun Miao  1 , Bin He, Yebin Jiang, Tatsuya Kobayashi, Maria A Sorocéanu, Jenny Zhao, Hanyi Su, Xinkang Tong, Norio Amizuka, Ajay Gupta, Harry K Genant, Henry M Kronenberg, David Goltzman, Andrew C Karaplis
Affiliations

Osteoblast-derived PTHrP is a potent endogenous bone anabolic agent that modifies the therapeutic efficacy of administered PTH 1-34

Dengshun Miao et al. J Clin Invest. 2005 Sep.

Abstract

Mice heterozygous for targeted disruption of Pthrp exhibit, by 3 months of age, diminished bone volume and skeletal microarchitectural changes indicative of advanced osteoporosis. Impaired bone formation arising from decreased BM precursor cell recruitment and increased apoptotic death of osteoblastic cells was identified as the underlying mechanism for low bone mass. The osteoporotic phenotype was recapitulated in mice with osteoblast-specific targeted disruption of Pthrp, generated using Cre-LoxP technology, and defective bone formation was reaffirmed as the underlying etiology. Daily administration of the 1-34 amino-terminal fragment of parathyroid hormone (PTH 1-34) to Pthrp+/- mice resulted in profound improvement in all parameters of skeletal microarchitecture, surpassing the improvement observed in treated WT littermates. These findings establish a pivotal role for osteoblast-derived PTH-related protein (PTHrP) as a potent endogenous bone anabolic factor that potentiates bone formation by altering osteoblast recruitment and survival and whose level of expression in the bone microenvironment influences the therapeutic efficacy of exogenous PTH 1-34.

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Figures

Figure 1
Figure 1
PTHrP haploinsufficiency causes osteopenia by decreasing bone formation. (A) Three-dimensional reconstruction from CT scans of distal femora from mice at 3 months of age. (B) Quantitative analysis of BV/TV. (C) Trabecular separation (Tb.Sp). (D) Degree of anisotropy (DA). (E) Structure model index (SMI). (F) Calcein (white arrowheads) and tetracycline (yellow arrowheads) labeling of trabecular bone (magnification, ×400). The distance between the 2 labels is used to calculate MAR, shown at right. (G) Red nuclear-stained apoptotic osteoblasts (arrows) and osteocytes (arrowheads) in the trabeculae were detected by TUNEL assay (magnification, ×400). The percentages of apoptotic osteoblasts and osteocytes (Apoptotic OB) are shown at right. (H) Ex vivo cultures of adherent BM cells induced to undergo osteogenic differentiation. Red staining represents alkaline phosphatase enzymatic activity, a marker of osteogenic differentiation. Quantitation of osteogenic colonies is shown at right. (I) TRAP stain for osteoclasts (magnification, ×200) and quantitation of osteoclast surface (Oc.S/BS). Data are shown as the mean ± SEM of 6 animals per group. *P < 0.05; **P < 0.01; and ***P < 0.001 for Pthrp+/– (black bars) versus Pthrp+/+ mice (white bars).
Figure 2
Figure 2
Generation of Pthrpflox/flox;creColI mice. (A) Decalcified paraffin sections stained histochemically for HPAP activity. HPAP activity was detected only in preosteoblasts and osteoblasts in Z/AP;creColI mice but not in Z/AP mice. (B) Genomic organization of WT and floxed Pthrp alleles as well as changes in the restriction enzyme pattern anticipated following digestion of tail-tip genomic DNA with BamHI and hybridization with a 0.65-kb SacI/XhoI genomic DNA fragment as probe (–). Arrowheads represent loxP sequences flanking exon 3. (C) Floxed Pthrp allele and Cre transgene detected by Southern blot analysis of tail-tip genomic DNA. The flox/flox–Cre-positive mice comprised the Pthrpflox/flox;creColI experimental group, while the Pthrpflox/flox mice were controls. (D) Decalcified paraffin sections immunostained for PTHrP. PTHrP immunoreactivity (red arrowheads) was seen in growth plate chondrocytes of Pthrpflox/flox;creColI mice and control mice but in preosteoblasts and osteoblasts only in control mice. Magnification, ×400.
Figure 3
Figure 3
Osteoblast-specific Pthrp ablation reduces BMD and trabecular bone volume. (A–C) Serum calcium (A), 1,25(OH)2D3 (B), and PTH (C) were measured as described in Methods. White bars, Pthrpflox/flox mice; black bars, Pthrpflox/flox;creColI mice. Data are shown as mean ± SEM. (D) Parathyroid gland histology was assessed from paraffin sections of thyroparathyroidal tissue stained with H&E. Magnification, ×200. (E) Faxitron radiographs of femur and tibia. (F) Three-dimensional reconstruction of the proximal tibiae from μCT scans. (G) Micrographs from undecalcified sections of the proximal end of tibia stained with the von Kossa procedure. Magnification, ×25. (H) BMD measurements at femurs and tibiae. (I–L) Quantitative histomorphometry for BV/TV (I), trabecular number (Tb.N) (J), trabecular thickness (Tb.Th) (K), and trabecular separation (L). Data shown represent mean ± SEM of 5–6 animals per group. *P < 0.05 and **P < 0.01 for Pthrpflox/flox;creColI (black bars) versus Pthrpflox/flox control mice (white bars).
Figure 4
Figure 4
Impaired osteoblastic bone formation in the absence of osteoblast-derived PTHrP. (A) Micrographs of the proximal ends of tibiae after double calcein labeling of the cortex (left 2 panels) and trabeculae (right 2 panels). (B) Micrographs from decalcified paraffin sections stained with H&E. Black arrowheads show osteoblastic cells. (C) Micrographs from undecalcified sections stained by the von Kossa procedure. White arrowheads show osteoids. (D) Red nuclear-stained apoptotic osteoblasts (arrows) and osteocytes (arrowheads) in the endosteum (left 2 panels) and trabeculae (right 2 panels) were detected by TUNEL assay. (E–J) Quantitative assessment by histomorphometric analysis of MAR at the cortex (E), MAR at trabeculae (F), osteoblast number/tissue area ratio (N.Ob/T.Ar) (G), osteoblast volume/bone surface ratio (Ob.S/BS) (H), osteoid volume/bone volume ratio (OV/BV) (I), and percentage of apoptotic osteoblasts (J). Data shown represent mean ± SEM of 5–6 animals per group. *P < 0.05, **P < 0.01, and ***P < 0.001 for Pthrpflox/flox;creColI (black bars) versus Pthrpflox/flox mice (white bars).
Figure 5
Figure 5
Formation of osteogenic colonies and osteoclasts in the absence of osteoblast-derived PTHrP. (A) Ex vivo primary BM cell cultures stained with methyl blue (top panels) or cytochemically for alkaline phosphatase (bottom panels). (B) Positive CFU-f and CFU-fALP numbers were counted manually and are presented as mean ± SEM of 3 animals per group. The experiment was performed twice with similar results. (C) Bone sections stained histochemically for TRAP activity. (D and E) Quantitative assessment by histomorphometric analysis of osteoclast number/total area ratio (N.Oc/T.Ar) (D) and osteoclast surface (E). Data shown represent mean ± SEM of 5–6 animals per group. **P < 0.01 and ***P < 0.001 for Pthrpflox/flox;creColI (black bars) versus Pthrpflox/flox mice (white bars).
Figure 6
Figure 6
Anabolic action of PTH 1–34 in Pthrp+/+ and Pthrp+/– mice. (A) Three-dimensional reconstruction of the distal femora from μCT scans after treatment with either vehicle (left panels) or PTH 1–34 (40 μg/kg/d) (right panels). (B) Micrographs of decalcified paraffin sections of vertebrae stained with H&E after treatment with either vehicle (left panels) or PTH 1–34 (right panels). Magnification, ×25. (C) Histomorphometric examination of double calcein labeling after treatment with either vehicle (left panels) or PTH 1–34 (right panels). Magnification, ×400. (D) Quantitative assessment of MAR. White bars, Pthrp+/+ mice; black bars, Pthrp+/– mice.
Figure 7
Figure 7
Data from quantitative assessment. (A) BV/TV. (B) Trabecular number. (C) Trabecular thickness. (D) Trabecular separation. (E) Trabecular connectivity (CD). (F) Degree of anisotropy. (G) Structure model index. (H) Cortical thickness (Ct.Th). Data shown represent mean ± SEM of 5–7 animals per group. White bars, Pthrp+/+ mice; black bars, Pthrp+/– mice.
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