The RANKL distal control region is required for the increase in RANKL expression, but not the bone loss, associated with hyperparathyroidism or lactation in adult mice

Abstract

Osteoclast-mediated bone resorption plays an essential role in calcium homeostasis and lactation. The cytokine receptor activator of nuclear factor κB ligand (RANKL) is one of a number of factors that controls the production, survival, and activity of osteoclasts. Calciotropic hormones, such as PTH, control RANKL transcription in part via an enhancer known as the distal control region (DCR), and mice lacking this enhancer have fewer osteoclasts under normal physiological conditions. Here, we have addressed the role of the DCR in situations in which activation of the PTH receptor is thought to stimulate bone resorption via elevation of RANKL expression. Dietary calcium deficiency stimulated RANKL expression in the bone of young (1 month old) wild-type, but not DCR knockout (KO), mice. Consistent with this, the cancellous bone loss and the increase in osteoclasts caused by dietary calcium deficiency were blunted in young KO mice. DCR deletion also prevented the increase in RANKL expression caused by dietary calcium deficiency in 6-month-old mice. However, the diet-induced bone loss was similar in wild-type and KO mice at this age. The increase in RANKL expression caused by lactation was also blunted in DCR KO mice, but lactation-induced bone loss was similar in both genotypes. These results demonstrate that, even though the DCR is required for the increase in RANKL expression associated with hyperparathyroidism or lactation, this increase is not required for the bone loss caused by these conditions in adult mice, suggesting that changes in other factors, such as osteoprotegerin or estrogen levels, play a dominant role.

Fig. 1.

Deletion of the DCR blunts cancellous bone loss induced by dietary calcium deficiency in growing mice. A–L, One-month-old WT and DCR KO mice were fed control or calcium-deficient diet for 3 (A) or 7 d (B–L). A and B, Quantitative RT-PCR of RANKL mRNA in L5 vertebrae of WT and KO mice after 3 (A) or 7 d (B) of experimental diet. C and D, Percent change in BMD of the spine (C) and femur (D) of WT or DCR KO mice, determined by DEXA. E–H, μCT analysis of bone volume over tissue volume (BV/TV) (E), trabecular spacing (Tb.Sp) (F), trabecular number (Tb.N) (G), and trabecular thickness (Tb.Th) (H) in L4 vertebrae. I–L, Histomorphometric analysis of bone area over tissue area (BA/TA) (I), trabecular spacing (Tb.Sp) (J), osteoclast number per bone perimeter (Oc.N/B.Pm) (K), and osteoclast perimeter per bone perimeter (Oc.Pm/B.Pm) (L) in L1–L3 vertebra. All quantitative RT-PCR values were normalized to the housekeeping gene ribosomal protein S2. BMD percent change and gene expression values represent the mean of 10–12 animals per group (both sexes), and μCT and histomorphometry values represent the mean of five to seven animals per group (females). All statistical comparisons were done with two-way ANOVA. *, P < 0.05 effect of diet within genotype. #, P < 0.05 effect of genotype within diet.

Fig. 2.

The difference in bone mass between WT and DCR KO increases with age. A–D, Femoral and vertebral BMD of male (A and B) and female (C and D) WT and DCR KO mice were measured serially from 1 to 12 months of age. The same cohort of animals was used for each time point. E and F, Bone volume per tissue volume (BV/TV) of L4 vertebrae and femoral cortical thickness (Ct.Th) were determined by μCT analysis in male (E) and female (F) mice. BMD values represent the mean of 12–19 mice per group, and μCT values represent the mean of four to eight animals per group. *, P < 0.05 vs. WT by Student's t test.

Fig. 3.

DCR deletion did not alter bone loss induced by dietary calcium deficiency in adult mice. Six-month-old WT and DCR KO female mice were fed control or calcium-deficient diet for 30 d. A and B, Percent change in BMD in the femur (A) and spine (B) was determined by comparison of DEXA at the start and end of 30 d of experimental diet. C and D, Bone volume over tissue volume (BV/TV) ratios of cancellous bone were determined by μCT analysis of femur (C) and L4 vertebrae (D). Values represent the mean of 10–12 animals per group. All statistical comparisons were done using two-way ANOVA. *, P < 0.05 effect of diet within genotype. #, P < 0.05 effect of genotype within diet.

Fig. 4.

The DCR is required for the increase in RANKL caused by dietary calcium deficiency. A and B, Quantitative RT-PCR analysis of RANKL mRNA levels in calvaria (A) and L5 vertebrae (B) of WT and DCR KO mice. C, sRANKL levels measured from blood plasma of WT and KO mice. D and E, Quantitative RT-PCR analysis of cathepsin K (CatK) (D) and OPG (E) mRNA levels in calvaria of WT and DCR KO mice. F, OPG protein levels measured from blood plasma of WT and KO mice. All quantitative RT-PCR values were normalized to ribosomal protein S2 mRNA levels and are the mean of 10–11 animals per group. Blood plasma protein values are the mean of 9–10 samples per group. All statistical comparisons were done using two-way ANOVA. *, P < 0.05 effect of diet within genotype; #, P < 0.05 effect of genotype within diet.

Fig. 5.

DCR deletion does not alter bone loss caused by lactation. Six-month-old WT and DCR KO females were mated with C57BL/6 males and then allowed to give birth and lactate for 12 d. A and B, Percent change in the BMD in the spine (A) and femur (B) of WT or DCR KO mice, determined by DEXA at d 4 and 12 of lactation. C and D, Bone volume over tissue volume (BV/TV) (C) and trabecular thickness (Tb.Th) (D) determined by μCT analysis of L4 vertebrae. E and F, Cortical thickness (Ct.Th) (E) and cortical porosity (F) determined by μCT analysis of the femur. BMD values represent the mean of 9–12 animals per group, and the μCT values are the mean of five to nine animals per group. All statistical comparisons were done using two-way ANOVA. *, P < 0.05 lactation vs. nulliparity within genotype; #, P < 0.05 WT vs. KO within lactation status.

Fig. 6.

DCR deletion blunts the lactation-induced increase in RANKL expression. A and B, Quantitative RT-PCR analysis of RANKL mRNA levels in calvaria (A) and L5 vertebrae (B) of WT and DCR KO mice. C, sRANKL levels measured from blood plasma of WT and KO mice. D and E, Quantitative RT-PCR analysis of cathepsin K (CatK) (D) and OPG (E) mRNA levels in calvaria of WT and KO mice. F, OPG protein levels measured from blood plasma of WT and KO mice. All mRNA levels are normalized to ribosomal protein S2 mRNA levels, and the values are the mean of five to nine animals per group. All statistical comparisons were done using two-way ANOVA. *, P < 0.05 lactation vs. nulliparity within genotype; #, P < 0.05 effect of genotype within lactation status.