Control of bone mass and remodeling by PTH receptor signaling in osteocytes

Abstract

Osteocytes, former osteoblasts buried within bone, are thought to orchestrate skeletal adaptation to mechanical stimuli. However, it remains unknown whether hormones control skeletal homeostasis through actions on osteocytes. Parathyroid hormone (PTH) stimulates bone remodeling and may cause bone loss or bone gain depending on the balance between bone resorption and formation. Herein, we demonstrate that transgenic mice expressing a constitutively active PTH receptor exclusively in osteocytes exhibit increased bone mass and bone remodeling, as well as reduced expression of the osteocyte-derived Wnt antagonist sclerostin, increased Wnt signaling, increased osteoclast and osteoblast number, and decreased osteoblast apoptosis. Deletion of the Wnt co-receptor LDL related receptor 5 (LRP5) attenuates the high bone mass phenotype but not the increase in bone remodeling induced by the transgene. These findings demonstrate that PTH receptor signaling in osteocytes increases bone mass and the rate of bone remodeling through LRP5-dependent and -independent mechanisms, respectively.

Figure 1. Generation of DMP1-caPTHR1 Transgenic Mice.

(A) Schematic representation of the DMP1-caPTHR1 transgene. The transgene contains the cDNA encoding the H223R constitutively active mutant of PTHR1 (caPTHR1) inserted downstream from a 12-kb DNA fragment containing 8 kb of the murine DMP1 5′-flanking region, first exon, first intron, and 17 bp of exon 2; a synthetic polyadenylation site was inserted downstream from the caPTHR1 sequence. Arrows indicate the sites recognized by primers used for genotyping. (B) Quantitative RT-PCR analysis of DMP1-caPTHR1 mRNA from the 5^th lumbar vertebrae (L5), tibia, and soft tissues of 10.5-week-old mice, normalized to the housekeeping gene ribosomal protein S2. Bars represent the mean±SD of 4 mice. (C) Expression of the human PTHR1, murine PTHR1, and Sost determined by quantitative RT-PCR in freshly isolated osteoblast-enriched (GFP−) and osteocyte-enriched (GFP+) cell preparations obtained from neonatal mice without (WT) or with (TG) the DMP1-caPTHR1 transgene.

Figure 2. DMP1-caPTHR1 Mice Exhibit High Bone Mass.

(A) Radiographs of 4-week-old female wild type (WT) and DMP1-caPTHR1 transgenic (TG) mice. (B) Femoral and spinal BMD in WT and TG female mice, measured at 4 wk intervals up to 24 wks of age. Symbols represent the mean±SD of 5 mice. * p<0.05 vs. WT mice at each time point. (C) Representative longitudinal and cross-sectional micro-CT images of femur, 4th lumbar vertebra, and calvaria obtained from 10.5-week-old female WT and TG mice. (D) Hematoxylin and eosin staining of tibial and (E) von Kossa staining of femoral bone sections from 10.5-week-old WT and TG mice.

Figure 3. DMP1-caPTHR1 Transgenic Mice Have Increased Bone Turnover.

(A–D) Histomorphometric measurements were determined in the distal femur, excluding the growth plate, of 3.5-week-old DMP1-caPTHR1 mice (TG) and wild type littermates (WT). Both sexes were included. Bone area (A), trabecular architecture (B), osteoblast perimeter, osteoclast perimeter, and osteocyte density (C), and quiescent surface (D) are shown. (E) Distal femoral cancellous bone of WT or TG mice viewed with epifluorescence to reveal calcein and alizarin labels or with polarized light with a rose quartz plate to reveal collagen architecture (×200). (F) Serum osteocalcin, serum CTX, and urinary DPD were measured in 3.5- to 5-week-old mice. (G) Osteoblast and osteocyte apoptosis measured in femoral sections from 3.5-week-old mice. (H) CFU-F and CFU-OB obtained from bone marrow cells of 4.5-week-old mice. Bars represent the mean±SD of 3–5 mice. * p<0.05 vs. WT mice.

Figure 4. Sost and Sclerostin Expression are Decreased and Wnt Signaling is Increased in DMP1-caPTHR1 Transgenic Mice.

(A) Quantitative RT-PCR analysis of Sost mRNA in tibia from 9-week-old DMP1-caPTHR1 mice and wild type littermates (n = 3). Both sexes were included. (B) Sclerostin protein levels were determined by Western blot analysis of lysates from the 6th lumbar vertebrae of 9-week-old wild type (WT) and DMP1-caPTHR1 (TG) mice. Each lane contains protein lysate from a single mouse. (C) Anti-sclerostin immunohistochemistry in ulnae sections from 10.5-week-old WT and TG mice. (D) β-galactosidase activity was measured in lysates of femurs obtained from 4.5-week-old TCF-βgal reporter mice with and without the DMP1-caPTHR1 transgene, and is expressed as relative luminescence units (RLU)/200 mg protein (n = 3–5 mice/group). Quantitative RT-PCR analysis of the indicated Wnt target genes (E), and OPG, RANKL, and M-CSF (F), in tibia from 8-week-old DMP1-caPTHR1 mice and wild type littermates. Bars represent the mean±SD of 3 mice. * p<0.05 vs. WT mice.

Figure 5. The High Bone Mass Phenotype of the DMP1-caPTHR1 Mice is Ameliorated in Mice Lacking the Wnt Co-Receptor LRP5.

(A) Serial analysis of BMD in the femur and spine of LRP5+/+ and LRP−/− mice, with and without the DMP1-caPTHR1 transgene. Both sexes were included. Bars represent the mean±SD of 3–5 mice/group. * p<0.05 vs. LRP5+/+ mice without the transgene. # p<0.05 vs. LRP+/+ mice without the transgene. (B) Von Kossa staining of femurs and lumbar vertebrae of 12-week-old LRP5+/+ and LRP−/− mice, with and without the DMP1-caPTHR1 transgene. (C) High resolution micro-CT scans of distal femora from LRP5+/+ and LRP−/− mice, with and without the DMP1-caPTHR1 transgene.

Figure 6. The High Bone Remodeling Phenotype of DMP1-caPTHR1 Mice does not Require LRP5.

(A) Osteocalcin and CTX measured in plasma from 12-week-old LRP5+/+ and LRP−/− mice, with and without the DMP1-caPTHR1 transgene. Both sexes were included. (B) Bone material density as determined by micro-CT analysis of femurs from LRP5+/+ and LRP−/− mice, with and without the DMP1-caPTHR1 transgene. Bars represent the mean±SD of 3–5 mice/group. * p<0.05 vs. LRP5+/+ mice without the transgene; # p<0.05 vs. LRP5+/+ mice with the transgene. (C) Representative images of histologic sections showing calcein and alizarin double labeling in femurs from LRP5+/+ and LRP−/− mice with and without the DMP1-caPTHR1 transgene.

Figure 7. PTHR1 Signaling in Osteocytes Leads to Increased Bone Mass and Remodeling via Distinct Mechanisms.

In the proposed model, PTHR1 signaling in osteocytes activates at least two distinct pathways: one leading to increased bone mass and the other leading to increased bone remodeling. Suppression of Sost/sclerostin and activation of LRP5 signaling increase osteoblast numbers and are required for the increase in bone mass. The elevation of bone resorption is independent of the Sclerostin/LRP5 pathway. The question mark indicates uncertainty of the cellular source of RANKL and M-CSF (osteocytes versus stromal/osteoblastic cells).