Skeletal recovery after weaning does not require PTHrP

Abstract

Mice lose 20% to 25% of trabecular bone mineral content (BMC) during lactation and restore it after weaning through unknown mechanisms. We found that tibial Pthrp mRNA expression was upregulated fivefold by 7 days after weaning versus end of lactation in wild-type (WT) mice. To determine whether parathyroid hormone-related protein (PTHrP) stimulates bone formation after weaning, we studied a conditional knockout in which PTHrP is deleted from preosteoblasts and osteoblasts by collagen I promoter-driven Cre (Cre(ColI) ). These mice are osteopenic as adults but have normal serum calcium, calcitriol, and parathyroid hormone (PTH). Pairs of Pthrp(flox/flox) ;Cre(ColI) (null) and WT;Cre(ColI) (WT) females were mated and studied through pregnancy, lactation, and 3 weeks of postweaning recovery. By end of lactation, both genotypes lost lumbar spine BMC: WT declined by 20.6% ± 3.3%, and null decreased by 22.5% ± 3.5% (p < .0001 versus baseline; p = NS between genotypes). During postweaning recovery, both restored BMC to baseline: WT to -3.6% ± 3.7% and null to 0.3% ± 3.7% (p = NS versus baseline or between genotypes). Similar loss and full recovery of BMC were seen at the whole body and hind limb. Histomorphometry confirmed that nulls had lower bone mass at baseline and that this was equal to the value achieved after weaning. Osteocalcin, propeptide of type 1 collagen (P1NP), and deoxypyridinoline increased equally during recovery in WT and null mice; PTH decreased and calcitriol increased equally; serum calcium was unchanged. Urine calcium increased during recovery but remained no different between genotypes. Although osteoblast-derived PTHrP is required to maintain adult bone mass and Pthrp mRNA upregulates in bone after weaning, it is not required for recovery of bone mass after lactation. The factors that stimulate postweaning bone formation remain unknown.

Fig. 1

Pthrp mRNA expression is upregulated in bone during postweaning recovery. Weaning was forced on day 12 of lactation, and skeletal mRNA was analyzed by real-time quantitative RT-PCR. PTHrP expression increased more than fivefold in bone through the first 7 days after weaning. Values were normalized to GAPDH and then expressed relative to the value on day 12 of lactation.

Fig. 2

obPthrp null and WT mice experience identical changes in BMC during pregnancy and lactation and after weaning. Relative changes in whole-body (A), spine (B), and hind limb (C) BMC versus baseline are depicted on day 18.5 of pregnancy, day 21 of lactation, and day 21 of postweaning recovery. The number of observations is indicated in parentheses.

Fig. 3

Bone turnover is increased after weaning. The bone markers osteocalcin (A), deoxypyridinoline (B), and P1NP (C) increased equally in WT and obPthrp null mice during postweaning recovery compared with the prepregnancy baseline. Depicted here are baseline, day 18.5 of pregnancy, day 21 of lactation, and day 7 of postweaning recovery. The most marked increase in bone resorption occurs during the first 10 days of lactation, but the time point assayed here was day 21 of lactation, when the pups were weaned. The number of observations is indicated in parentheses.

Fig. 4

Flux in PTH and calcitriol during reproductive cycles. (A) PTH normally becomes suppressed during pregnancy and lactation but rises to baseline during postweaning recovery. This pattern was noted in WT and obPthrp null mice, although the changes were not statistically significant. (B) Calcitriol tripled during pregnancy, as expected, and then declined during recovery but remained double the baseline value. No significant differences were seen between WT and obPthrp null mice. Time points depicted are baseline, day 18.5 of pregnancy, day 21 of lactation, and day 7 of postweaning recovery. The number of observations is indicated in parentheses.

Fig. 5

Flux in urinary calcium and phosphorus during reproductive cycles. (A) Urinary calcium normally increases during pregnancy and decreases during lactation; the fall during lactation was statistically significant. (B) Urinary phosphorus decreased nonsignificantly during pregnancy but increased significantly during lactation as a consequence of enhanced bone resorption and possibly PTHrP-stimulated phosphaturia. In all panels, there were no significant differences between WT and obPthrp null mice. Time points depicted are baseline, day 18.5 of pregnancy, day 21 of lactation, and day 7 of postweaning recovery. The number of observations is indicated in parentheses.