Parathyroid hormone receptor signaling induces bone resorption in the adult skeleton by directly regulating the RANKL gene in osteocytes

Abstract

PTH upregulates the expression of the receptor activator of nuclear factor κB ligand (Rankl) in cells of the osteoblastic lineage, but the precise differentiation stage of the PTH target cell responsible for RANKL-mediated stimulation of bone resorption remains undefined. We report that constitutive activation of PTH receptor signaling only in osteocytes in transgenic mice (DMP1-caPTHR1) was sufficient to increase Rankl expression and bone resorption. Resorption in DMP1-caPTHR1 mice crossed with mice lacking the distal control region regulated by PTH in the Rankl gene (DCR(-/-)) was similar to DMP1-caPTHR1 mice at 1 month of age, but progressively declined to reach values undistinguishable from wild-type (WT) mice at 5 months of age. Moreover, DMP1-caPTHR1 mice exhibited low tissue material density and increased serum alkaline phosphatase activity at 5 month of age, and these indices of high remodeling were partially and totally corrected in compound DMP1-caPTHR1;DCR(-/-) male mice, and less affected in female mice. Rankl expression in bones from DMP1-caPTHR1 mice was elevated at both 1 and 5 months of age, whereas it was high, similar to DMP1-caPTHR1 mice at 1 month, but low, similar to WT levels at 5 months in compound mice. Moreover, PTH increased Rankl and decreased Sost and Opg expression in ex vivo bone organ cultures established from WT mice, but only regulated Sost and Opg expression in cultures from DCR(-/-) mice. PTH also increased RANKL expression in osteocyte-containing primary cultures of calvarial cells, in isolated murine osteocytes, and in WT but not in DCR(-/-) osteocyte-enriched bones. Thus, PTH upregulates Rankl expression in osteocytes in vitro, ex vivo and in vivo, and resorption induced by PTH receptor signaling in the adult skeleton requires direct regulation of the Rankl gene in osteocytes.

Figure 1.

Deletion of the DCR progressively corrects the high resorption induced by PTH receptor signaling in osteocytes in male and female mice. A, Longitudinal analysis of CTX for the male cohort from 1 to 5 months. Symbols represent the means ± SD of 7 to 16 mice per group. #, P < .05 for DMP1-caPTHR1 vs DMP1-caPTHR1;DCR^−/− CTX levels at 1 and 5 months. Bars represent means ± SD of 7 to 16 mice per group. *, P < .05 DMP1-caPTHR1 or DMP1-caPTHR1;DCR^−/− vs WT and DCR^−/−, respectively, by two-way ANOVA. B, Representative images of cross-sections of the femoral midshaft of 5-month-old males stained for TRAPase/toluidine blue. Bars indicate 50 μm. C, Longitudinal analysis of CTX for the female cohort from 1 to 5 months. Symbols represent the means ± SD of 7 to 16 mice per group. #, P < .05 DMP1-caPTHR1 vs DMP1-caPTHR1;DCR^−/− CTX levels at 1 and 5 months. Bars represent means ± SD of 7 to 16 mice per group. *, P < .05 DMP1-caPTHR1 or DMP1-caPTHR1;DCR^−/− vs WT and DCR^−/−, respectively; #, P < .05 for conditions connected by the lines, by two-way ANOVA.

Figure 2.

Removal of the DCR does not affect the increase in BMD exhibited by DMP1-caPTHR1 mice. Longitudinal analysis of total, spinal, and femoral BMD in both males (A) and females (B) at 1-month intervals up to 5 months of age. Symbols are means ± SD (n = 7–19 mice per group). No significant differences were detected among phenotypes.

Figure 3.

Removal of the DCR blunts the increased bone remodeling induced by PTH receptor activation in osteocytes. A, Representative micro-CT images of distal femur and femoral midshaft of 5-month-old male mice. B, Representative images of unstained cross-sections of the femoral midshaft. Arrows point at the endocortical surface. C, Alkaline phosphatase was measured in plasma from 5-month-old male and female mice for all groups. Bars represent means ± SD of 7 to 11 male mice and 10 to 15 female mice per group. D, Bone material density was determined by micro-CT analysis of femoral bone from 5-month-old mice. Bars represent the means ± SD of 4 to 7 male mice and 4 female mice per group. *, P < .05 DMP1-caPTHR1 or DMP1-caPTHR1;DCR^−/− vs WT and DCR^−/−, respectively; #, P < .05 for conditions connected by the lines, by two-way ANOVA.

Figure 4.

Deletion of the DCR corrects the elevated expression of Rankl and M-Csf in adult, but not in young, DMP1-caPTHR1 mice. A–C, Quantitative PCR analysis of mRNA transcripts in calvariae from 1- and 5-month-old male mice. Bars represent means ± SD of 3 to 5 mice per group. D, Gene expression in calvaria from 5-month-old female mice. Bars represent means ± SD of 5 mice per group. Results are expressed relative to the housekeeping gene ribosomal protein S2 (ChoB). *, P < .05 DMP1-caPTHR1 or DMP1-caPTHR1;DCR^−/− vs WT and DCR^−/−, respectively, by two-way ANOVA.

Figure 5.

PTH receptor signaling modulates Rankl expression in osteocytes in vitro and ex vivo through direct regulation of the Rankl gene. The expression of the indicated genes was measured by quantitative PCR. Results are expressed relative to Gapdh. A and B, Ex vivo bone organ cultures were cultured overnight and treated with vehicle or PTH for 4 hours. Bars represent mean ± SD of 3 to 5 mice per genotype. *, P < .05 DMP1-caPTHR1 or DMP1-caPTHR1;DCR^−/− vs WT and DCR^−/−, respectively; #, P < .05 for conditions connected by the lines, by two-way ANOVA. C, Primary cultures of calvaria cells obtained from newborn C57BL/6 mice were treated with vehicle (veh), PTH, or DBA for 4 hours. Bars represent mean ± SD of 5 independent wells per treatment. *, P < .05 vs vehicle-treated cultures, by one-way ANOVA. D, Primary osteoblasts (OB) and osteocytes (OT) were isolated by FACS, and the expression of the indicated genes was quantified immediately after sorting. Bars represent mean ± SD of 2 independent wells and duplicate measurements per treatment. *, P < .05 PTH vs vehicle-treated cultures by two-way ANOVA; #, P < .05 for OT vs OB, by two-way ANOVA (for Rankl) or t test (for Pthr1).

Figure 6.

Osteocytic PTH receptor-driven resorption is controlled by the DCR of the Rankl gene in the mature skeleton. PTHR1 signaling through cAMP acts directly on the DCR to control Rankl expression and resorption in the mature murine skeleton. Stimulation of Rankl expression and resorption in the growing skeleton might result from increased gp130 cytokine signaling through signal transducer and activator of transcription 3 (STAT3) response elements or from increased 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) mediated by the vitamin D₃ receptor (VDR), or both. PKA, protein kinase A; CREB, cAMP response element-binding protein.