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. 2001 Feb;107(3):277-86.
doi: 10.1172/JCI11296.

Activated parathyroid hormone/parathyroid hormone-related protein receptor in osteoblastic cells differentially affects cortical and trabecular bone

L M Calvi  1 , N A Sims, J L Hunzelman, M C Knight, A Giovannetti, J M Saxton, H M Kronenberg, R Baron, E Schipani
Affiliations

Activated parathyroid hormone/parathyroid hormone-related protein receptor in osteoblastic cells differentially affects cortical and trabecular bone

L M Calvi et al. J Clin Invest. 2001 Feb.

Abstract

Parathyroid hormone (PTH), an important regulator of calcium homeostasis, targets most of its complex actions in bone to cells of the osteoblast lineage. Furthermore, PTH is known to stimulate osteoclastogenesis indirectly through activation of osteoblastic cells. To assess the role of the PTH/PTH-related protein receptor (PPR) in mediating the diverse actions of PTH on bone in vivo, we generated mice that express, in cells of the osteoblastic lineage, one of the constitutively active receptors described in Jansen's metaphyseal chondrodysplasia. In these transgenic mice, osteoblastic function was increased in the trabecular and endosteal compartments, whereas it was decreased in the periosteum. In trabecular bone of the transgenic mice, there was an increase in osteoblast precursors, as well as in mature osteoblasts. Osteoblastic expression of the constitutively active PPR induced a dramatic increase in osteoclast number in both trabecular and compact bone in transgenic animals. The net effect of these actions was a substantial increase in trabecular bone volume and a decrease in cortical bone thickness of the long bones. These findings, for the first time to our knowledge, identify the PPR as a crucial mediator of both bone-forming and bone-resorbing actions of PTH, and they underline the complexity and heterogeneity of the osteoblast population and/or their regulatory microenvironment.

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Figures

Figure 1
Figure 1
Transgene construct and its expression. (a) Shown is the scheme of the transgene construct with the location of probes A and B for Southern blot analysis of genomic DNA. Locations of the translation initiation codon (ATG), the receptor mutation (H223R), and the stop codon (Stop) in the cDNA encoding the human PPR mutant, HKrk-H223R, are seen. Restriction sites for the enzymes HindIII, PvuII, and ScaI are also indicated. Oligonucleotide S was used as probe for Southern blot analysis of RT-PCR products, oligonucleotides S1 and A were used for RT-PCR of total RNA, and oligonucleotides S2 and A1 were used to generate the DT7 probe. (b) Southern blot analysis of the RT-PCR products with probe S: RT-PCR products from line not expressing the transgene (lanes 1 and 6), CL1 (lanes 2, 3, 7, and 8), and CL2 (lanes 4 and 9), with negative control in lane 5 and –RT controls in lanes 6–9. (c–h) In situ hybridization with the 35S-labeled DT7 cRNA in serial section of decalcified proximal tibia of newborn CL2 mouse (c and d),and a wild-type littermate (g and h). In the high-magnification images from 2-week-old CL2 mutant (e and f), the arrow indicates the periosteal surface. (i) In situ hybridization with the 35S-labeled PPR cRNA in a section of decalcified proximal tibia of a newborn wild-type mouse. All sections were counterstained with hematoxylin and eosin; bright-field (c, e, g) and dark-field (d, f, h, i) views are shown.
Figure 2
Figure 2
Histology and histomorphometry of trabecular bone. (a–f) Histologic sections of undecalcified proximal tibia of 2-week-old (a–c) and 6-week-old (d–f) wild-type (a and d), CL1 (b and e), and CL2 (c and f) mice stained by the method of Von Kossa. (g–j) Histologic sections, stained with hematoxylin and eosin, of decalcified proximal tibia from 12-week-old wild-type (g) and CL2 (h) littermates. In the high-power images from the proximal tibia of 12-week-old wild-type (i) and CL2 transgenic (j) littermates, the metaphyseal area is shown. (k–p) Histomorphometric analysis of wild-type (light blue), CL1 (magenta), and CL2 (dark blue) littermates. Ages of animals are indicated on the x-axis. AP < 0.05, BP < 0.01, CP < 0.005. Error bars represent the SEM.
Figure 3
Figure 3
In situ hybridization analysis of trabecular bone. In situ hybridization with the 35S-labeled alkaline phosphatase (a, b, g, l), collagen I (c, h, m), collagenase 3 (d, i, n), osteopontin (e, j, o), and osteocalcin (f, k, p) cRNAs in serial sections of decalcified proximal tibia from 2-week-old CL2 mouse. Higher-magnification images of the trabecular area are shown (g–p). The sections were counterstained with hematoxylin and eosin; bright-field (a, l–p) and dark-field (b–k) views are shown.
Figure 4
Figure 4
Osteoclast number and function. (a–d) In situ hybridization with the 35S-labeled TRAP cRNA in serial sections of decalcified proximal tibia of 2-week-old wild-type (a and b) and CL2 (c and d) littermates. The sections were counterstained with hematoxylin and eosin; bright-field (a and c) and dark-field (b and d) views are shown. (e and f) High-power (×200) light microscopy of enzymatic staining for TRAP activity in sections of decalcified proximal tibia from a 2-week-old CL2 mouse, focusing on the metaphyseal trabecular bone (e) and the diaphyseal cortical bone (f); the arrow indicates one of the TRAP-positive multinucleated cells. Sections were counterstained with methyl green.
Figure 5
Figure 5
Analysis of cortical bone. (a–d) Histologic sections of cortical bone from tibial mid-diaphysis of 8-week-old wild-type (a) and CL1 transgenic (b) littermates and 12-week-old wild-type (c) and CL2 transgenic (d) littermates. (e and f) Histomorphometric analysis performed in 12-week-old wild-type (light blue) and CL2 (dark blue) littermates. AP < 0.05, BP < 0.01. Error bars represent the SEM. MAR, mineral apposition rate. (g–r) High-power (×200) light microscopy images of the mid-diaphysis of in situ hybridization with the 35S-labeled alkaline phosphatase (g–j), collagen I (k–n), and osteocalcin (o–r) cRNAs in serial sections of decalcified proximal tibia of 2-week-old wild-type (g, h, k, l, o, p) and CL2 (i, j, m, n, q, r) mice; sections were counterstained with hematoxylin and eosin. Bright-field (g, i, k, m, o, q) and dark-field (h, j, l, n, p, r) views are shown. The arrow indicates the periosteal surface.
Figure 6
Figure 6
Skull histology and histomorphometry. (a and b) Sections of skulls from 12-week-old wild-type (a) and CL2 (b) littermates, stained with the method of Von Kossa. Arrows show the endosteal surfaces. (c–e) Histomorphometric analysis of the skull from wild-type (light blue) and CL2 (dark blue) littermates at 12 weeks of age. AP < 0.05, BP < 0.01. Error bars represent the SEM.
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