Lactating Ctcgrp nulls lose twice the normal bone mineral content due to fewer osteoblasts and more osteoclasts, whereas bone mass is fully restored after weaning in association with up-regulation of Wnt signaling and other novel genes.

The maternal skeleton resorbs during lactation to provide calcium to milk and the lost mineral content is restored after weaning. The changes are particularly marked in Ctcgrp null mice, which lose 50% of spine mineral content during lactation but restore it fully. The known calciotropic hormones are not required for skeletal recovery to occur; therefore, unknown factors that stimulate bone formation may be responsible. We hypothesized that the genes responsible for regulating postweaning bone formation are differentially regulated in bone or marrow, and this regulation may be more marked in Ctcgrp null mice. We confirmed that Ctcgrp null mice had twice as many osteoclasts and 30-40% fewer osteoblasts as compared with wild-type mice during lactation but no deficit in osteoblast numbers after weaning. Genome-wide microarray analyses on tibial RNA showed differential expression of 729 genes in wild-type mice at day 7 after weaning vs prepregnancy, whereas the same comparison in Ctcgrp null mice revealed only 283 genes. Down-regulation of Wnt family inhibitors, Sost and Dkk1, and inhibition of Mef2c, a sclerostin stimulator, were observed. Ctsk, a gene expressed during osteoclast differentiation, and Igfbp2, which stimulates bone resorption, were inhibited. Differential regulation of genes involved in energy use was compatible with a net increase in bone formation. The most marked changes occurred in genes not previously associated with bone metabolism. In conclusion, the postlactation skeleton shows dynamic activity with more than 700 genes differentially expressed. Some of these genes are likely to promote bone formation during postweaning by stimulating the proliferation and activity of osteoblasts, inhibiting osteoclasts, and increasing energy use.

Skeletal recovery after weaning does not require PTHrP.

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.

Calcitonin plays a critical role in regulating skeletal mineral metabolism during lactation.

The maternal skeleton rapidly demineralizes during lactation to provide calcium to milk, responding to the stimuli of estrogen deficiency and mammary-secreted PTH-related protein. We used calcitonin/calcitonin gene-related peptide-alpha (Ctcgrp) null mice to determine whether calcitonin also modulates lactational mineral metabolism. During 21 d of lactation, spine bone mineral content dropped 53.6% in Ctcgrp nulls vs. 23.6% in wild-type (WT) siblings (P < 0.0002). After weaning, bone mineral content returned fully to baseline in 18.1 d in Ctcgrp null vs. 13.1 d in WT (P < 0.01) mice. Daily treatment with salmon calcitonin from the onset of lactation normalized the losses in Ctcgrp null mice, whereas calcitonin gene-related peptide-alpha or vehicle was without effect. Compared with WT, Ctcgrp null mice had increased circulating levels of PTH and up-regulation of mammary gland PTH-related protein mRNA. In addition, lactation caused the Ctcgrp null skeleton to undergo more trabecular thinning and increased trabecular separation compared with WT. Our studies confirm that an important physiological role of calcitonin is to protect the maternal skeleton against excessive resorption and attendant fragility during lactation and reveal that the postweaning skeleton has the remarkable ability to rapidly recover even from losses of over 50% of skeletal mineral content.

Ablation of calcitonin/calcitonin gene-related peptide-alpha impairs fetal magnesium but not calcium homeostasis.

We used the calcitonin/calcitonin gene-related peptide (CGRP)-alpha gene knockout model (Ct/Cgrp null) to determine whether calcitonin and CGRPalpha are required for normal fetal mineral homeostasis and placental calcium transfer. Heterozygous (Ct/Cgrp(+/-)) and Ct/Cgrp null females were mated to Ct/Cgrp(+/-) males. One or two days before term, blood was collected from mothers and fetuses and analyzed for ionized Ca, Mg, P, parathyroid hormone (PTH), and calcitonin. Amniotic fluid was collected for Ca, Mg, and P. To quantify skeletal mineral content, fetuses were reduced to ash, dissolved in nitric acid, and analyzed by atomic absorption spectroscopy for total Ca and Mg. Placental transfer of (45)Ca at 5 min was assessed. Ct/Cgrp null mothers had significantly fewer viable fetuses in utero compared with Ct/Cgrp(+/-) and wild-type mothers. Fetal serum Ca, P, and PTH did not differ by genotype, but serum Mg was significantly reduced in null fetuses. Placental transfer of (45)Ca at 5 min was normal. The calcium content of the fetal skeleton was normal; however, total Mg content was reduced in Ct/Cgrp null skeletons obtained from Ct/Cgrp null mothers. In summary, maternal absence of calcitonin and CGRPalpha reduced the number of viable fetuses. Fetal absence of calcitonin and CGRPalpha selectively reduced serum and skeletal magnesium content but did not alter ionized calcium, placental calcium transfer, and skeletal calcium content. These findings indicate that calcitonin and CGRPalpha are not needed for normal fetal calcium metabolism but may regulate aspects of fetal Mg metabolism.