Effect of calcium-deficient diet on bone turnover. A, Representative micrographs of proximal tibiae; B, diaphyseal cortical bone from both Wt and D/D mice fed a low-calcium diet or a corresponding control diet for 3 wk after weaning, stained with von Kossa for mineral, hematoxylin and eosin (H&E) for histology, and Masson-Goldner trichrome for bone matrix, as indicated. Both groups of mice on the low-calcium diet showed a similar dramatic loss of bone mineral and cortical bone mass, but H&E stain demonstrated a striking difference (indicated by insets) in metaphyseal trabecular bone turnover between Wt and D/D mice on the low-calcium diet. C, Histomorphometric measurements of diaphyseal cortical bone thickness and metaphyseal trabecular bone volume in 6-wk-ld Wt and D/D mice fed low-calcium and control diet, as indicated. Error bars represent se (n = 6); *, P < 0.05 vs. control diet; #, P < 0.05 vs. Wt.

DSEL mice on the low-calcium diet exhibited strikingly curtailed peritrabecular stromal cell accumulation, new bone formation, and hypertrophic expansion. A, Representative micrographs of proximal tibial metaphyseal trabecular bone from mice fed a low-calcium diet or a corresponding control diet for 3 wk after weaning, stained with hematoxylin and eosin (H&E) for histology (×20 magnification), showing expanded hypertrophic chondrocytes (as indicated by the black bracket) and accumulation of peritrabecular fibroblast-like stromal cells (indicated by arrows) in Wt but not in D/D mice fed the low-calcium diet, and stained with Masson-Goldner trichrome (×40 magnification), demonstrating massive disorganized newly formed undermineralized bone, shown in red, in Wt but not in D/D mice fed the low-calcium diet. B, Measurements of peritrabecular fibrosis. Fb.V/TV, Fibrosis volume/total volume. *, P < 0.05 vs. control diet; n = 6. C, Osteopontin in situ in proximal tibiae on the low-calcium diet for 3 wk.

DSEL mice with PTH infusion exhibited curtailed peritrabecular stromal cell responses and bone formation. A, Representative micrographs of proximal tibia from 4-wk-old mice receiving continuous infusion of hPTH (80 μg/kg · d) or vehicle through sc implantation of Alzet mini-osmotic pumps for 2 wk, stained with hematoxylin and eosin (H&E) for histology (×40 magnification), showing dramatic accumulation of peritrabecular fibroblast-like stromal cells (indicated by arrows) in Wt but not in D/D mice, and stained with Masson-Goldner trichrome and von Kossa (×40 magnification) demonstrating massive disorganized bone formation in Wt but not in D/D mice. B, Serum P1NP, CTX, intact PTH(1-84) (PTH), total calcium, and phosphorus (Pi) were measured in Wt and D/D mice after infusion with vehicle (white bars) and PTH (black bars). Error bars represent se (n = 6). a, P < 0.05 vs. vehicle; b, P < 0.05 vs. Wt PTH; c, P < 0.05 vs. Wt vehicle.

Effect of PTH on apoptosis and differentiation in primary osteoblastic cells. A, In vitro osteoblast apoptosis was quantified by TUNEL assay. Primary osteoblasts cultured in an eight-well slide chamber, were treated with vehicle or hPTH(1-34) (100 nm) for 24 h, and then in situ fluorescein (TUNEL, green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) stain were performed. TUNEL-positive cells were counted as apoptotic cells (percent, as shown in bar graph). Data are represented as mean ± se (two wells per slide in three independent experiments). *, P < 0.05 vs. vehicle (t test). B, Effect of PTH on osteoblast differentiation in vitro. Primary osteoblastic cells isolated from 6-wk-old tibiae were cultured in six-well or 12-well plates with α-MEM containing 10% FBS, and after confluence, cells were cultured with differentiation medium and treated continuously (freshly adding PTH every 48 h) or intermittently (adding PTH for 4 h in every 48 h) with 100 nm hPTH(1-34) for 4–6 wk. Mineralization was assessed by von Kossa stain and calcium (Ca) content (bar graph) was measured in acid extracts of the cultures (bar graph). ALP activity was determined by stain and also measured enzymatically (bar graph) (28).

PTHR signaling and bone mass in DSEL mice. A, Dose-dependent stimulation of cAMP by PTH in DSEL-derived primary osteoblastic cells. Primary osteoblastic cells isolated from Wt (white bars) and D/D (black bars) mice were cultured in 24-well plates, and after confluence, cells were incubated with cAMP assay buffer containing hPTH(1-34) (∼0–1000 nm) or forskolin (10 μm) for 20 min before cAMP measurement. B, PTH fails to induce intracellular calcium response in DSEL-derived primary osteoblastic cells. Primary osteoblastic cells (white bars for Wt cells and black bars for D/D cells) were cultured in 96-well plates, and after confluence, cells were incubated with Fluo-4 direct calcium buffer containing hPTH (∼0–1000 nm), ionomycin (Ionom) (10 μm), PMA (10 nm), or forskolin (Fsk) (10 μm) for 40 min before fluorescence signal measurement. Data are expressed as relative fluorescence units (RFU). C, No PLC activation by PTH in DSEL-derived osteoblastic cells. Primary osteoblastic cells from Wt and D/D mice were cultured in six-well plates. At 80% confluence, cells were labeled with [³H]myoinositol for 48 h and then stimulated with hPTH (1000 nm) (black bars) or vehicle (white bars) for 40 min in the presence of 20 mm LiCl. The cellular content of radioactive IP3 was determined. Values are mean ± sd of triplicate wells. *, P < 0.05 vs. vehicle. Each experiment was repeated twice with similar results. D, Representative micrographs of plastic sections of tibiae from sex-matched Wt and DSEL homozygous (D/D) male mice at the age of 10 wk, stained with von Kossa. E, μCT analysis of 10-wk-old distal femur from both male and female Wt (white bars) and D/D (black bars) mice. F, Serum P1NP and CTX were measured in both Wt and D/D mice at the age of 10 wk. Error bars in E and F represent se (n = 8). *, P < 0.05 vs. Wt mice. BV/TV, Bone volume/total volume; Tb. N, trabecular number; Tb. Sp, trabecular spacing.

Decreased colony formation and proliferation in DSEL-derived primary bone and bone marrow cells. A, Decreased colony formation in DSEL-derived bone marrow cells. Bone marrow cells from 4-wk-old tibiae on either a low-calcium diet (Ca diet) or a control diet for 1 wk were cultured in12-well plates for 14 d. The cultured plates were stained with methylene blue for total number of colonies (CFU-F) or with ALP staining solution to count positive colonies for ALP (CFU-ALP). Counts of colonies were performed blind on coded plates. *, P < 0.05 vs. Wt control diet; #, P < 0.05 vs. Wt low-calcium diet (n = 4). B, In vitro osteoblast proliferation. Primary osteoblasts were treated with vehicle (Veh), 100 nm hPTH(1-34) (PTH), 100 nm [G¹,R¹⁹]hPTH(1-28) (1-28), or 10 ng/ml basic fibroblast growht factor (bFGF) for 24 h and labeled with BrdU for 2 h. In situ anti-BrdU fluorescein (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) stain were performed and BrdU-positive cells were counted, as shown in bar graph (two wells per treatment in three independent experiments). *, P < 0.05 vs. vehicle. C and D, Real-time PCR detection of cyclin D1 mRNA expression in primary osteoblastic cells isolated from Wt and D/D mice (C) or from D/D mice (D). Primary osteoblasts were treated with 100 nm hPTH(1-34) (PTH) or [G¹,R¹⁹]hPTH(1-28) (1-28) (C) or with 10 nm PMA and 100 μm 8-bromo-cAMP (8Brom) (D) for 0–24 h, as indicated. Transcript expression of the cultured cells was determined by quantitative RT-PCR with primers specific for cyclin D1. Data are expressed as percent basal levels, and similar results were obtained in three independent experiments.