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. 1999 Feb;103(4):517-24.
doi: 10.1172/JCI5481.

Bone resorption induced by parathyroid hormone is strikingly diminished in collagenase-resistant mutant mice

W Zhao  1 , M H Byrne, B F Boyce, S M Krane
Affiliations

Bone resorption induced by parathyroid hormone is strikingly diminished in collagenase-resistant mutant mice

W Zhao et al. J Clin Invest. 1999 Feb.

Abstract

Parathyroid hormone (PTH) stimulates bone resorption by acting directly on osteoblasts/stromal cells and then indirectly to increase differentiation and function of osteoclasts. PTH acting on osteoblasts/stromal cells increases collagenase gene transcription and synthesis. To assess the role of collagenase in the bone resorptive actions of PTH, we used mice homozygous (r/r) for a targeted mutation (r) in Col1a1 that are resistant to collagenase cleavage of type I collagen. Human PTH(1-34) was injected subcutaneously over the hemicalvariae in wild-type (+/+) or r/r mice four times daily for three days. Osteoclast numbers, the size of the bone marrow spaces and periosteal proliferation were increased in calvariae from PTH-treated +/+ mice, whereas in r/r mice, PTH-induced bone resorption responses were minimal. The r/r mice were not resistant to other skeletal effects of PTH because abundant interstitial collagenase mRNA was detected in the calvarial periosteum of PTH-treated, but not vehicle-treated, r/r and +/+ mice. Calcemic responses, 0.5-10 hours after intraperitoneal injection of PTH, were blunted in r/r mice versus +/+ mice. Thus, collagenase cleavage of type I collagen is necessary for PTH induction of osteoclastic bone resorption.

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Figures

Figure 1
Figure 1
Paraffin sections stained with H&E staining of calvarial bone from +/+ and r/r mice injected subcutaneously over the hemicalvariae with vehicle or PTH. Shown are representative photomicrographs of sections of calvarial bone that illustrate resorptive responses to PTH of +/+ and r/r mice. Bones from vehicle-injected +/+ mice showed a normal smooth surface of bone marrow spaces and no osteoclasts. Bones from +/+ mice injected with PTH demonstrated marked resorption with increased osteoclasts (arrows) in cavities with irregular surfaces (measured as bone resorption area and illustrated in Fig. 3). In contrast, the appearance of bones from the PTH-treated r/r mice was similar to that of vehicle-treated r/r mice, and few osteoclastic resorption spaces were found. Scale bar, 0.05 mm. H&E, hematoxylin and eosin; PTH, parathyroid hormone.
Figure 2
Figure 2
Presence of osteoclast-like cells, identified by TRAP staining, in calvarial bone from +/+ mice injected subcutaneously over the hemicalvariae with PTH. The identification of osteoclasts in the PTH-treated +/+ calvariae was confirmed using TRAP staining. (a) The section stained with H&E. (b) An adjacent section stained with TRAP. Arrows indicate multinucleated osteoclasts in intracalvarial resorption spaces. Scale bar, 0.05 mm. TRAP, tartrate-resistant acid protein.
Figure 3
Figure 3
Changes in calvarial bone area and osteoclast number in 4-week-old +/+ and r/r mice injected subcutaneously over the hemicalvariae with vehicle (Veh) or PTH. Black bars (means ± SEM) indicate the bone marrow space area (total resorption area), expressed as a percentage of the total bone area in a standard length of calvarial sections. Clear bars (means ± SEM) indicate the number of osteoclasts in the total bone area (i.e., per mm2). The number of mice examined are indicated in parentheses. The bone resorption area and the number of osteoclasts/mm2 were both significantly increased after PTH injection in the +/+ mice (P < 0.005), but not in the r/r mice. In contrast, resorption in response to PTH was markedly reduced, and only occasional osteoclasts were identified in calvarial bone from PTH-treated r/r mice.
Figure 4
Figure 4
Changes in calvarial bone resorption area in +/+ and r/r mice of different ages injected subcutaneously over the hemicalvariae with vehicle (Veh) or PTH. Only total bone resorption is shown as means ± SEM. The data labeled EXPT. 1 are from the study shown in Fig. 3, using 4-week-old mice. The number of mice examined are indicated in parentheses. The age of the mice in EXPT. 2 was 4–6 weeks, and in EXPT. 3 and EXPT. 4, 9–12 months. In each of the four individual experiments, the general pattern of bone resorption in response to PTH was similar. Bone resorption in response to PTH was markedly reduced in the r/r mice compared with the +/+ mice.
Figure 5
Figure 5
In situ hybridization, using collagenase and 92-kDa gelatinase riboprobes, of the resorption areas in calvarial bone from +/+ mice injected subcutaneously over the hemicalvariae with PTH. Shown are calvariae from +/+ mice injected with PTH. As can be seen in sections visualized using dark field and bright field, the 92-kDa gelatinase was strongly expressed in osteoclasts but weakly expressed in nonosteoclastic surrounding cells. In contrast, collagenase was expressed in cells other than osteoclasts and the signal was most intense in cells adjacent to the osteoclasts within the intracortical resorption spaces. Arrows indicate periosteneum. Scale bar, 0.01 mm.
Figure 6
Figure 6
In situ hybridization, using a collagenase riboprobe, of calvarial bone from +/+ and r/r mice injected subcutaneously over the hemicalvariae with vehicle or PTH. The outer (subcutaneous) side is shown. Sections were hybridized with the antisense collagenase riboprobe and were visualized using bright field (top) and dark field (bottom). Arrows indicate the periosteum. No hybridization with the collagenase riboprobe was detected in the periosteum in calvariae from vehicle-treated +/+ mice or vehicle-treated r/r mice. In calvariae from both +/+ and r/r mice, levels of collagenase mRNA in periosteal osteoblasts were markedly increased after PTH injection. No signal was observed in samples hybridized with the collagenase sense riboprobe (not shown). Scale bar, 0.05 mm.
Figure 7
Figure 7
Changes in blood [Ca2+] in +/+ and r/r mice injected intraperitoneally with vehicle or PTH. The calcemic response after a single intraperitoneal injection of vehicle or 15 μg PTH in +/+ and r/r mice is shown as described for the first calcemia experiment. The change in blood ionized calcium levels, Δ blood [Ca2+], at intervals from 0.5 to 10 h after PTH injection (shown as means ± SEM) is depicted on the vertical axis. Peak levels of Δ blood [Ca2+] were observed within the first 1–3 h after PTH injection in the +/+ mice (filled circles). As described in the text, peak levels of Δ blood [Ca2+] after PTH injection in the r/r mice were significantly lower than in the +/+ mice (filled squares).
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