M-CSF neutralization and egr-1 deficiency prevent ovariectomy-induced bone loss

Abstract

Increased stromal cell production of M-CSF, an event caused by enhanced phosphorylation of the nuclear protein Egr-1, is central to the mechanism by which estrogen (E2) deficiency upregulates osteoclast (OC) formation. However, the contribution of enhanced M-CSF production to the bone loss induced by E2 deficiency remains to be determined. We found that treatment with an Ab that neutralizes M-CSF in vivo completely prevents the rise in OC number, the increase in bone resorption, and the resulting bone loss induced by ovariectomy (ovx). We also found that adult, intact Egr-1-deficient mice, a strain characterized by maximally stimulated stromal cell production of M-CSF, exhibit increased bone resorption and decreased bone mass. In these mice, treatment with anti-M-CSF Ab restored normal levels of bone resorption, thus confirming that increased M-CSF production accounts for the remodeling abnormalities of Egr-1-deficient mice. Consistent with the failure of ovx to further increase M-CSF production in Egr-1-deficient mice, ovx neither increased bone resorption further, nor caused bone loss in these animals. In summary, the data demonstrate that E2 deficiency induces M-CSF production via an Egr-1-dependent mechanism that is central to the pathogenesis of ovx-induced bone loss. Thus, Egr-1 and M-CSF are critical mediators of the bone sparing effects of E2 in vivo.

Figure 1

Effect of serum from 5A1 Ab–treated mice on M-CSF–induced monocyte proliferation. Results (mean + SEM) are expressed as percent of M-CSF–induced proliferation. Sera (100 μL) obtained at the end of the treatment period from ovx mice treated with either 5A1 Ab or irrelevant Ab and fresh 5A1 Ab (200 ng in 100 μL) were serially diluted (1:2) and added to CD11b⁺ cells seeded in triplicate in 96-well plates (final volume 200 μL per well). Recombinant murine M-CSF (25 ng/mL) was then added to each well. Cell proliferation was measured after a 3-day incubation, as described in Methods. ^AP < 0.05, compared with the other groups.

Figure 2

Treatment with 5A1 Ab prevents ovx-induced bone loss. Results (mean ± SEM) are expressed as percent of change from baseline. Mice 4 months of age were ovx or sham-operated. Ovx mice were treated with either the anti–M-CSF Ab 5A1, an isotype-matched irrelevant Ab, or E2 for the first 4 weeks after surgery. Sham-operated mice were left untreated. In vivo measurements of rear-limb BMD were carried out by DEXA as described in Methods before surgery and 2 and 4 weeks after surgery (n = 10 mice per group). Baseline BMD values were as follows: Sham, 62.5 ± 1.0 mg/cm²; ovx-irrelevant Ab, 61.9 ± 0.6 mg/cm²; ovx E2, 62.8 ± 0.9 mg/cm²; ovx 5A1 Ab, 63.6 ± 1.0 mg/cm². There was no significant difference for baseline BMD among groups. ^AP < 0.05 compared with baseline and with any other group.

Figure 3

Treatment with 5A1 Ab prevents the increase in DPD excretion induced by ovx. Results (mean ± SEM) are expressed as percent of change from baseline. Mice were treated as described in Figure 1 (n = 10 mice per group). Baseline DPD values were as follows: sham, 18.6 ± 0.4 nmol/mmol Cr; ovx irrelevant Ab, 18.8 ± 0.4 nmol/mmol Cr; ovx E2, 17.2 ± 0.2 nmol/mmol Cr; ovx 5A1 Ab, 19.9 ± 0.4 nmol/mmol Cr. There was no significant difference for baseline DPD excretion among groups. ^AP < 0.05 compared with baseline and with any other group.

Figure 4

(a) BMD, (b) DPD excretion, and (c) osteocalcin levels in adult Egr-1^–/– mice and control WT littermates (mean ± SEM). Measurements were carried out in samples (n = 30 per group) from 4-month-old intact mice. ^AP < 0.05 compared with WT mice.

Figure 5

Treatment with 5A1 Ab decreases DPD excretion in Egr-1–deficient mice. Intact adult WT and Egr-1^–/– mice were treated with either 5A1 Ab or irrelevant Ab for 4 weeks as described above. Results (mean + SEM) are expressed as absolute values (n = 5 mice per group). ^AP < 0.05 compared with both groups of WT mice.

Figure 6

Egr-1–deficient mice are protected against ovx-induced bone loss. Results (mean ± SEM) are expressed as percent of change from baseline. Thirty Egr-1^–/– mice and 30 control WT littermates were either ovx or sham-operated. Ovx mice were treated with either vehicle or E2 for 4 weeks. Sham-operated mice were left untreated (n = 10 mice per group). In vivo measurements of rear-limb BMD were carried out as described in Figure 2. ^AP < 0.05 compared with baseline and to any other group.

Figure 7

Ovx fails to increase DPD excretion in Egr-1^–/– mice. Results (mean + SEM) are expressed as percent of change from baseline. Mice were treated as described in Figure 6 (n = 10 mice per group). ^AP < 0.05 compared with baseline and to any other group.