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A major part of age-related bone loss in women is related to estrogen deficiency in the postmenopausal period.1 Estrogen deficiency leads to increased bone turnover and increased osteoclastic bone resorption owing to enhanced production of a number of proinflammatory and osteoclastogenic cytokines.2
Delta-like 1/preadipocyte factor 1 (Dlk1/Pref1) is an imprinted gene encoding a transmembrane protein that belongs to the epidermal growth factor (EGF)–like repeat-containing family of proteins.3 The Dlk1 protein exists both as a membrane-bound protein and as a soluble factor (known as fetal antigen 1 [FA1]) released from the cell membrane through proteolytic cleavage by ADAM 17.4 The importance of Dlk1 in normal skeletal physiology has been demonstrated by the presence of growth disturbances as well as skeletal abnormalities in gene-modified mice.5–7 Also, in the human syndrome of maternal uniparental disomy (matUPD14), where Dlk1/Pref1 is silent, patients exhibit obesity, hypotonia, premature puberty, macrocephaly, short stature, and small hands,8, 9 whereas patients with paternal uniparental disomy (patUPD14), where Dlk1/Pref1 is overexpressed, exhibit a narrow thorax with abnormally curved ribs, facial dysmorphism, and mild hypoplasia of the ilia.10
We have reported previously that circulating s-Dlk1/FA1 functions as an endocrine factor that regulates bone mass in adult mice.11 Recently, we have reported that a high level of circulating s-Dlk1/FA1 was associated with bone loss in mice caused by enhancing bone resorption and inhibition of bone formation. In addition, Dlk1 deficient mice were protected from ovariectomy (OVX)–induced bone loss.7 To further investigate the role of Dlk1/FA1 in human physiology, we examined the effects of estrogen (E) deficiency and E replacement on serum levels of Dlk1/FA1 and its correlation with other known bone turnover markers.
Materials and Methods
Human cohort 1: Healthy pre- and postmenopausal women
Fifty healthy premenopausal women (aged 30 to 40 years, mean age 35 years) and 50 healthy untreated postmenopausal women (aged 48 to 73 years, mean 59 years) were included. All premenopausal women had regular cyclic menses. All postmenopausal women were amenorrhoeic for at least 5 years. A fasting serum sample was collected before 9 am and stored at a temperature below –70 °C until analyzed. The institution's ethical review board approved the study.
Human cohort 2: Postmenopausal women receiving estrogen-replacement therapy
Study subjects were recruited from an age-stratified random sample of Rochester, MN, USA.12 The Mayo Institutional Review Board approved the study, and informed consent was obtained from all subjects. The cohort included 166 postmenopausal women consisting of two groups: The estrogen-replacement therapy (ERT) group consisted of 83 postmenopausal women; mean (SD) age and body mass index (BMI) were 63.3 (10.1) and 27.2 (5.8), respectively, and the control group consisted of 83 subjects who did not receive ERT, selective estrogen receptor modulators (SERMs), or bisphosphonates; mean (SD) age and BMI were 63.4 (10.1) and 27.2 (54.8), respectively. The estrogen regimen in the study subjects included either estrogen and progesterone combination therapy or estrogen alone (if hysterectomized) for varying durations of 1 year to more than 10 years and two subjects who had been on estrogen for less than 1 year. There are four women (three ERT women and one control) who have a serum creatinine level greater than 1.2 mg/dL. Fasting-state serum samples were obtained and were stored at –70 °C until analyzed. We did not specifically exclude women based on vitamin D levels.
Measurements of serum Dlk1/FA1 and biochemical markers of bone turnover
Quantification of human Dlk1/FA1 (hFA1) was performed as described previously using a sandwich ELISA technique.13 Both the intra- and interassay coefficients of variation (CVs, %) were less than 5%. The bone-resorption marker cross-linked C-telopeptide (CTX-I; epitope EKAHDGGR), which is cleaved and released from the C-terminal of type 1 collagen,14 was measured using a commercial kit: CrossLaps ELISA (Nordic Bioscience, Herlev, Denmark); intra- and interassay CVs were 7% and 9%, respectively. Human serum osteocalcin was measured by a commercial radioimmunoassay ELISA-OST-NAT that recognizes both the N- and C-termini of the osteocalcin molecule (Cis BioInternational, Bedford, MA, USA).15 The intra- and interassay CVs were less than 6%.
The differences in Dlk1/FA1 levels in the human case-control study were assessed by the Mann-Whitney test. The correlations between Dlk1/FA1 and serum bone markers were examined using linear regression analysis and the Pearson correlation coefficient (r).
We measured s-Dlk1/FA1 and s-CTX-1 in a cohort of healthy premenopausal (n = 50) and postmenopausal (n = 50) women. Both markers were significantly elevated in the postmenopausal women compared with the premenopausal women (Table 1), and s-Dlk1/FA1 was positively correlated with s-CTX-1 (Fig. 1A). To investigate the ability of ERT to inhibit the observed increase in s-Dlk1/FA1 in postmenopausal women, we compared s-Dlk1/FA1 levels in untreated and E-treated postmenopausal women (n = 83 per group). Table 1 demonstrates that s-Dlk1/FA1, s-CTX-1, and s-osteocalcin all were decreased significantly in postmenopausal women receiving ERT. In addition, the two bone turnover markers were correlated with each other (r = 0.7581, p < 0.0001), and each of the bone markers was positively correlated with the s-Dlk1/FA1 (Fig. 1B, C).
Table 1. Comparison of the Serum Levels of Dlk1/FA1, CTX-1, or Osteocalcin Between GROUPS Within the Two Human Cohorts
Cohort 1 (N= 100)
23.0 (20.6, 25.5)
0.298 (0.262, 0339)
41.9 (37.0, 46.8)
0.456 (0.403, 0.509)
Cohort 2 (N= 166)
Postmenopausal women + ERT
29.9 (27.2, 32.7)
0.388 (0.321, 0.455)
16.4 (14.5, 18.2)
41.5 (37.9, 45.1)
0.647 (0.573, 0722)
22.9 (20.6, 25.3)
In this study we have demonstrated that E deficiency in humans is associated with increased serum levels of Dlk1/FA1 that were normalized by ERT. Additionally, changes in s-Dlk1/FA1 were positively correlated with changes in serum bone turnover markers, suggesting a role in mediating E action on the skeleton. These results were obtained from two population cohorts. Cohort 1 revealed an association between E status and s-FA1, and cohort 2 was an intervention study that provided a causal relationship between E levels and s-FA1.
The range of s-FA1 in normal adults is 12.3 to 46.6 ng/mL (age range 19 to 60 years),16 and its levels reflect a balance between production and clearance rate. During development, Dlk1/FA1 is expressed widely in various organs; however, in adults, Dlk1/FA1 is produced by neuroendocrine organs, for example, adrenal glands, pituitary gland, β-cells in the pancreas, male and female gonads, and the hypothalamus, as well as other distinct nuclei in the CNS.17 s-Dlk1/FA1 is cleared through the kidneys.16 s-Dlk1/FA1 is elevated in several human diseases, including neurofibromatosis,18 small cell lung cancer,19 anorexia nervosa,20 and renal failure.16
The effects of estrogen on s-Dlk1/FA1 can take place at several levels: transcription/translation, proteolysis/shedding, or clearance. Since estrogen receptors are widely distributed in different tissues, including the neuroendocrine tissues,21 it is plausible that estrogen affects the production of s-Dlk1/FA1. We have identified several possible cell types that produce Dlk1/FA1 in the bone marrow and are known targets for E action, including osteoprogenitors/stromal cells, preadipocytes, B cells, and T cells.22 Thus our working hypothesis is that E deficiency results in upregulation of the level of Dlk1/FA1 in the bone microenvironment, which will, in turn, activate the NF-κB signaling pathway and increase the production of proinflammatory cytokines, and that will lead to an increase in bone turnover, enhance bone resorption, and inhibit bone formation resulting in bone loss.7, 11 In support of this hypothesis is the finding of partial protection of ovariectomy-induced bone loss in Dlk1-deficient mice.7 It is also possible that E causes changes in s-Dlk1/FA1 indirectly through other hormonal changes. Menopause and ERT lead to changes serum growth hormone,23 which is known to lead to changes in s-Dlk1/FA1.24
In conclusion, targeting Dlk1/FA1 is potentially a novel approach to preventing the deleterious effects of estrogen deficiency on the skeleton.
All the authors state that they have no conflicts of interest.
BMA, ABJ, and BS contributed equally to this article. This work was supported by grants from the Lundbeck Foundation, the Novo Nordisk Foundation, the local government of southern Denmark, and the NIH (AR027065). The technical help of Ms Bianca Jørgensen and the advice of Dr Charlotte H Jensen regarding the ELISA assay are acknowledged.
Authors' roles: BMA conceived and performed experiments, developed the project, and wrote the manuscript. ABJ analyzed data, and helped with writing manuscript. BS conducted CTX-1 and osteocalcin measurements.
BMA, ABJ, and BS contributed equally to this article.