Mammary-specific deletion of parathyroid hormone-related protein preserves bone mass during lactation

Abstract

Large amounts of calcium are transferred to offspring by milk. This demand results in negative calcium balance in lactating mothers and is associated with rapid bone loss. The mechanisms of bone loss during lactation are only partly understood. Several studies have suggested that parathyroid hormone-related protein (PTHrP) might be secreted into the circulation by the lactating mammary gland and regulate bone turnover during lactation. Because mammary development fails in the absence of PTHrP, conventional PTHrP knockout mice cannot be used to address this possibility. To examine this hypothesis, we therefore used mice carrying a beta-lactoglobulin promoter-driven Cre transgene, one null PTHrP allele, and one floxed PTHrP allele. Expression of Cre specifically in mammary epithelial cells during late pregnancy and lactation resulted in efficient deletion of the PTHrP gene; mammary gland PTHrP mRNA and milk PTHrP protein were almost completely absent. Removal of PTHrP from the lactating mammary glands resulted in reductions in levels of circulating PTHrP and 1,25-dihydroxy vitamin D and urinary cAMP. In addition, bone turnover was reduced and bone loss during lactation was attenuated. We conclude that during lactation mammary epithelial cells are a source of circulating PTHrP that promotes bone loss by increasing rates of bone resorption.

Figure 1

(a) Ribonuclease protection analysis of PTHrP and cyclophilin transcripts in RNA from yeast (lane 1) and mammary glands from virgin PTHrP^lox/– (lane 2) and BLG-Cre/PTHrP^lox/– (lane 3) mice shows that PTHrP is normally expressed at low levels; therefore significant recombination has not yet taken place. (b) Ribonuclease protection of PTHrP and cyclophilin in RNA from mammary glands of lactating BLG-Cre/PTHrP^lox/– (lane 2) and PTHrP^lox/– (lane 3) mice. Yeast RNA (lane 1) was a negative control. The efficiency of BLG-Cre–mediated recombination is highlighted by the essentially complete lack of PTHrP RNA in the BLG-Cre/PTHrP^lox/– mammary glands. (c) PTHrP protein was undetectable (12 of 14) or drastically reduced (2 of 14) in milk from BLG-Cre/PTHrP^lox/– mice (mean 0.06 ± 0.04 nM, n = 14) compared with littermate PTHrP^lox/– controls (16.4 ± 4.1 nM, n = 9) as a result of the mammary-specific targeting of PTHrP gene deletion.

Figure 2

Mammary gland whole mounts stained with carmine aluminum (a and b) and histological sections stained with hematoxylin and eosin (c and d) from lactating PTHrP^lox/– mice (a and c) and BLG-Cre/PTHrP^lox/– mice (b and d). The scale bar in b represents 200 μm, and the scale bar in d represents 5 μm. BLG-Cre/PTHrP^lox/– and control mammary glands appeared to be typical of lactating mammary tissue.

Figure 3

Calcium metabolism in BLG-Cre/PTHrP^lox/– mice. Circulating PTHrP(1–34) levels (a) were significantly lower (P < 0.05) during lactation in BLG-Cre/PTHrP^lox/– mice (0.73 ± 0.13 pM, n = 7) than in PTHrP^lox/– controls (1.18 ± 0.19 pM, n = 3). Circulating calcium (b) and PTH (c) concentrations were not significantly different between PTHrP^lox/– and BLG-Cre/PTHrP^lox/– mice during lactation. Probably as a result of the reduced circulating PTHrP, urinary cAMP levels (d) and circulating 1,25-dihydroxy vitamin D concentrations (e) were also significantly reduced. Milk calcium (f) was not significantly different in BLG-Cre/PTHrP^lox/– mice and controls. Bars represent the mean values of 3–12 samples. PTH concentrations were 3.8 ± 1.5 pM in PTHrP^lox/– controls and 3.7 ± 0.9 pM in BLG-Cre/PTHrP^lox/– mice. Although statistically insignificant, both plasma calcium and milk calcium are slightly lower in BLG-Cre/PTHrP^lox/– mice (8.8 ± 0.2 mg/dl and 406.7 ± 39.4 mg/dl, n = 14) than in controls (9.0 ± 0.3 mg/dl and 471 ± 78.5 mg/dl, n = 9). Urinary cAMP was 33.9 ± 0.6 μg/mmol creatinine in PTHrP^lox/– controls (n = 7) and 19.3 ± 0.8 μg/mmol creatinine in BLG-Cre/PTHrP^lox/– mice (n = 11), while 1,25-dihydroxy vitamin D levels were 55.9 ± 5.9 pg/ml in PTHrP^lox/– mice (n = 3) and 43.5 ± 2.2 pg/ml in BLG-Cre/PTHrP^lox/– mice (n = 6).

Figure 4

Biochemical markers of bone turnover were lower in lactating BLG-Cre/PTHrP^lox/– mice. (a) Urine CTx levels were significantly lower (P < 0.05) in BLG-Cre/PTHrP^lox/– mice (3.98 ± 0.82 μg/mmol creatinine, n = 8) than in controls (6.83 ± 0.29 μg/mmol creatinine, n = 7). (b) Osteocalcin concentrations in plasma were lower in BLG-Cre/PTHrP^lox/– mice than controls (48.4 ± 5.1 ng/ml, n = 14, and 66.8 ± 12.5 ng/ml, n = 9, respectively), but these values were not significantly different.

Figure 5

Bone density was measured in vivo at days 4 and 11 of lactation by DEXA. At day 4, the vertebral BMD of BLG-Cre/PTHrP^lox/– mice (n = 6) and controls (n = 5) was 0.05294 ± 0.002043 g/cm² and 0.05290 ± 0.002030 g/cm². By day 11 of lactation, the BMD of the control mice had fallen to 0.04368 ± 0.001386 g/cm², whereas the BMD of the BLG-Cre/PTHrP^lox/– mice was 0.04786 ± 0.001512 g/cm². Therefore, the BLG-Cre/PTHrP^lox/– mice lost significantly less bone mass during a 7-day time period during lactation than did controls (P < 0.01).