Increased production of IL-7 uncouples bone formation from bone resorption during estrogen deficiency

Abstract

Postmenopausal bone loss stems from the inability of osteoblastic activity to match the increase in osteoclastic bone resorption induced by estrogen deficiency. However, the mechanism that uncouples osteoblast from osteoclast activities remains unexplained. We show that ovariectomy enhances the production of the osteoclastogenic cytokine IL-7, and that its neutralization in vivo prevents ovariectomy-induced bone loss. Surprisingly, serum osteocalcin levels, a biochemical marker of bone formation, suggested that the bone-sparing effects of IL-7 neutralization were due not only to inhibition of bone resorption, but also to stimulation of bone formation. Consistent with these data, addition of IL-7 to neonatal calvarial organ cultures blocked new bone formation, and injection of IL-7 into mice in vivo inhibited bone formation as measured by calcein incorporation into long bones. The antianabolic effects of IL-7 were consistent with an observed downregulation of the osteoblast-specific transcription factor core-binding factor alpha1/Runx2. Thus, because it targets both the osteoclast and the osteoblast pathways, IL-7 is central to the altered bone turnover characteristic of estrogen deficiency.

Figure 1

E2 deficiency upregulates IL-7 mRNA and protein concentrations in murine bone marrow cells in vivo. (a) Levels of IL-7 mRNA were analyzed by semiquantitative RT-PCR using whole murine bone marrow isolated from E2-deficient ovx mice and E2-replete mice (sham-operated, and ovx receiving E2 replacement) 2 weeks after surgery (n = 4 mice per group). RNA concentrations were internally controlled using the housekeeping gene GAPDH. IL-7 was evaluated at 30 PCR cycles and GAPDH at 25. IL-7 was confirmed to be below saturation up to 35 cycles and GAPDH up to 30 cycles (data not shown). Data are representative of two independent experiments. (b) Quantitation of IL-7 mRNA. IL-7 mRNA bands in a were quantitated by densitometry using NIH Image and normalized for GAPDH concentrations. (c) IL-7 protein concentrations in the marrow compartments of E2-replete and E2-deficient mice were measured by ELISA in cell-free bone marrow serum immediately following isolation of the marrow, as described in Methods. Data represent average ± SEM of four mice per group. Data points were measured in quadruplicate. *P ≤ 0.05 (ANOVA).

Figure 2

IL-7 neutralization in vivo prevents bone loss following ovx. (a) Mice were sham-operated (filled squares) or ovx-operated (open diamonds), with one additional ovx group receiving E2 replacement (open circles). Another group of ovx mice was injected intraperitoneally with 1 mg of anti–IL-7 neutralizing antibody three times per week (open squares), and one group of ovx mice received irrelevant isotype-matched (IgG2b) antibody (filled triangles). BMD (mg/cm²) was measured by DEXA at base line (0) and at 2 and 4 weeks after ovx. Data are shown as average ± SEM. *P ≤ 0.01 compared with sham-operated mice and ovx mice receiving E2 replacement; ⁺P ≤ 0.02 compared with ovx mice injected with anti–IL-7 antibody (ANOVA; n = 6 mice per group). (b) The urinary concentration of DPD was measured at 4 weeks after surgery by ELISA, and data were corrected for urinary excretion using creatinine. Data represent average percent change from sham-operated ± SEM. Samples were measured in triplicate. *P ≤ 0.05 compared with sham-operated mice (ANOVA; n = 6 mice per group). (c) Serum OCN was measured at sacrifice by RIA. Data represent average percent change from sham-operated ± SEM. Samples were measured in triplicate. *P ≤ 0.05 compared with sham-operated mice; ⁺P ≤ 0.05 compared with ovx-operated mice receiving irrelevant IgG2b antibody (ANOVA; n = 6 mice per group).

Figure 3

IL-7 inhibits both basal and BMP2-stimulated bone formation in neonatal calvarial organ cultures in vitro. Neonatal calvaria (2 days old) were cultured for 2 weeks in the presence or absence of IL-7 (50 ng/ml) and/or BMP2 (50 ng/ml). Calvaria were stained with hematoxylin and eosin and photographed at ×100 magnification. Dark pink bands indicate old bone, while light pink bands represent newly formed bone. Data are representative of two independent experiments.

Figure 4

IL-7 inhibits bone formation in mice in vivo. Mice were injected intraperitoneally with IL-7 (10 μg/kg body weight) or vehicle (PBS plus 0.1% BSA) for 30 days and pulsed with calcein 7 and 3 days prior to sacrifice. Tibiae were plastic-embedded, sectioned, and examined by fluorescence microscopy. (a) Representative calcein fluorescence bands from four different mice are shown for each group. Bands (1 mm in length) were photographed at comparable anatomic positions at the mid-diaphysis of each bone at ×100 magnification. (b) Fluorescence intensity of the entire area of each of the four individual panels shown in a, for both vehicle- and IL-7–treated groups, was quantitated by densitometry using NIH Image. Data represent the average OD of four panels per group ± SEM.

Figure 5

IL-7 suppresses the transactivation of a promoter driven by six OSE2 elements and blunts Cbfa1/Runx2 promoter activity in ROS 17/2.8 osteoblastic cells. (a) p6OSE2-luc, a luciferase reporter construct driven by six tandem copies of OSE2 fused to a minimal OCN promoter, and p6OSE2mut-luc, a control construct containing six inactive OSE2 sites, were transfected into ROS 17/2.8 osteoblastic cells, and luciferase activity was assessed at 24 hours in unstimulated cells and cells stimulated with IL-7 (10 ng/ml). Data are shown as average ± SD of triplicate cultures and are representative of three independent experiments. *P < 0.05 (t test). (b) pGL3–Cbfa1/Runx2, a luciferase reporter construct containing the distal OB-specific Cbfa1/Runx2 promoter (–620 bp), or empty vector (pGL3-basic) was transfected into ROS 17/2.8 osteoblastic cells, and luciferase activity was assessed at 24 hours in unstimulated cells and cells stimulated with IL-7 (10 ng/ml). The data are shown as average ± SD of triplicate cultures and are representative of three independent experiments. *P < 0.05 (t test).

Figure 6

IL-7 inhibits OPG production in ROS 17/2.8 cells. (a) Using semiquantitative RT-PCR, we measured OPG mRNA at 24 hours in ROS 17/2.8 cells stimulated with vehicle (PBS plus 0.1% BSA) or IL-7 (10 ng/ml). Data are representative of two independent experiments. (b) OPG mRNA bands in a were quantitated by densitometry and normalized against GAPDH. (c) OPG protein production was measured by Western blot at 24 hours in primary calvarial OBs stimulated with vehicle (PBS plus 0.1% BSA) or IL-7 (10 ng/ml). Data are representative of two independent experiments. (d) OPG protein concentrations in c were quantitated by densitometry.