Tob deficiency superenhances osteoblastic activity after ovariectomy to block estrogen deficiency-induced osteoporosis

Abstract

Tob (transducer of erbB2) is a member of antiproliferative family proteins and acts as a bone morphogenic protein inhibitor as well as a suppressor of proliferation in T cells, which have been implicated in postmenopausal bone loss. To determine the effect of Tob deficiency on estrogen deficiency-induced bone loss, we analyzed bone metabolism after ovariectomy or sham operation in Tob-deficient mice. Ovariectomy in WT mice decreased trabecular bone volume and bone mineral density (BMD) as expected. In Tob-deficient mice, ovariectomy reduced bone volume and BMD. However, even after ovariectomy, both trabecular bone volume and BMD levels in Tob-deficient bone were comparable to those in sham-operated WT bones. Bone formation parameters (mineral apposition rate and bone formation rate) in the ovariectomized Tob-deficient mice were significantly higher than those in the ovariectomized WT mice. In contrast, the ovariectomy-induced increase in the bone resorption parameters, osteoclast surface, and osteoclast number was similar between Tob-deficient mice and WT mice. Furthermore, in ex vivo nodule formation assay, ovariectomy-induced enhancement of nodule formation was significantly higher in the bone marrow cells from Tob-deficient mice than in the bone marrow cells from ovariectomized WT mice. Both Tob and estrogen signalings converge at bone morphogenic protein activation of alkaline phosphatase and GCCG-reporter gene expression in osteoblasts, revealing interaction between the two signals. These data indicate that Tob deficiency prevents ovariectomy-induced bone loss through the superenhancement of osteoblastic activities in bone and that this results in further augmentation in the bone formation rate and the mineral apposition rate after ovariectomy in vivo.

Fig. 1.

Uterine weight after ovariectomy in Tob-deficient mice. Shown is uterine weight in sham-operated (SHAM) and OVX WT mice and Tob-deficient (TOB KO) mice. Wet uterus was weighed at the end of the experiments. Ovariectomy significantly decreased the uterine weight in both WT mice and Tob-deficient mice. The reduction levels in uterus weight after OVX were comparable between WT mice and Tob-deficient mice. Asterisks indicate statistical significance.

Fig. 2.

Tob deficiency preserves bone volume and BMD even after ovariectomy-induced reduction. (A) μCT analysis of the femora of mice. WT or Tob-deficient (TOB KO) mice were either ovariectomized (OVX) or sham-operated (SHAM). Trabecular bone patterns in both WT mice and Tob-deficient mice were altered to be more sparse after ovariectomy. (B) μCT-based quantification of the BV/TV in sham-operated (SHAM) or OVX WT and Tob-deficient (TOB KO) mice. BV/TV within the area indicated by a rectangle was quantified as described in Materials and Methods. WT mice and Tob-deficient mice were reduced after ovariectomy compared with that in the sham-operated mice. Although bone volume in Tob-deficient mice was reduced after ovariectomy compared with sham-operated Tob-deficient mice, the final levels were still comparable to those in the sham-operated WT mice. (C) μCT-based quantification of the cortical bone volume in sham-operated (SHAM) or OVX WT and Tob-deficient (TOB KO) mice. The cortical bone area was measured by using μCT sections in a plane perpendicular to the long axis within the mid-diaphysis. (D) BMD of the whole femora of sham-operated (SHAM) or OVX WT and Tob-deficient (TOB KO) mice was measured by using a small animal dual-energy x-ray absorptiometry apparatus. Similar to BV/TV, BMD levels in OVX Tob-deficient mice were comparable to those in sham-operated WT mice and were higher than those in OVX WT mice.

Fig. 3.

Tob deficiency further enhances the ovariectomy-induced increase in bone formation in vivo. Bone formation parameters were measured in histomorphometric analysis of tibia. MAR (A) and BFR (B) were determined based on calcein labeling in sham-operated (SHAM) or OVX WT and Tob-deficient (TOB KO) mice. Calcein was injected at 4 mg/kg 4 and 2 days before killing. Measurement was conducted in the secondary spongiosa in tibiae. MAR and BFR were enhanced by ovariectomy in WT mice. Basal levels of MAR and BFR in Tob-deficient mice were comparable to those in OVX WT mice. However, ovariectomy further enhanced MAR and BFR levels in Tob-deficient mice compared to WT mice. ^*, P < 0.05.

Fig. 4.

Tob deficiency did not alter the ovariectomy-induced increase in the levels of bone resorption parameters. Bone resorption parameters were obtained based on histomorphometric analysis of bone. Oc.S/BS (A) and N.Oc/BS (B) in sham-operated (SHAM) or OVX WT and Tob-deficient (TOB KO) mice were examined. Oc.S/BS and N.Oc/BS levels were increased in WT mice and Tob-deficient mice after ovariectomy, and these levels were similar between WT mice and Tob-deficient mice. (C) Urinary Dpyd levels in sham-operated (SHAM) or OVX WT and Tob-deficient (TOB KO) mice. Similar to Oc.S/BS and N.Oc/BS levels, the levels of urinary Dpyd in OVX Tob-deficient mice were similar to those in OVX WT mice. ^*, P < 0.05.

Fig. 5.

Tob deficiency further enhances the ovariectomy-induced increase in nodule formation in vitro. Functional characterization of the bone marrow cells obtained from WT mice and Tob-deficient mice was conducted after OVX using cultures of these cells. (A) Mineralized nodule formation in the cultures of bone marrow cells from sham-operated (SHAM) or OVX WT mice and Tob-deficient (TOB KO) mice. The cells were cultured in presence of ascorbic acid and β-glycerophosphate for 21 days. OVX-induced enhancement in nodule formation was significantly more in the bone marrow cells from Tob-deficient mice than that in WT mice. (B) Osteoclast formation in the cultures of bone marrow cells from sham-operated (SHAM) or OVX WT mice and Tob-deficient (TOB KO) mice. The cells were treated with vitamin D and dexamethasone for 14 days. Enhancement in osteoclast development in cultures induced by ovariectomy was similar between Tob-deficient mice and WT mice. ^*, P < 0.05.

Fig. 6.

Tob deficiency further augments the ovariectomy-induced enhancement in the expression of genes encoding osteoblast phenotype-related proteins. mRNA expression of the genes encoding bone formation-related proteins and bone resorption-related proteins was examined in bone marrow tissue. (A) Ovariectomy-induced enhancement in the levels of mRNA expression of bone formation-related genes in the bone marrow in Tob-deficient mice was more than that in WT mice. (B) Ovariectomy enhanced mRNA expression levels of bone resorption-related genes encoding RANK, RANK ligand, and osteoprotegerin (OPG) in bone marrow. The enhancement levels were similar between WT mice and Tob-deficient mice.

Fig. 7.

BMP signaling is enhanced by the absence of exogenous estrogen and Tob in osteoblasts. (A) Convergence of Tob and estrogen signaling in the regulation of phenotype (ALP) expression in osteoblasts. Smad1, estrogen receptor, and Tob expression vectors were cotransfected into ROS17/2.8 osteoblastic cells, which were then exposed to BMP2 or estrogen or were further transfected with Tob expression vector. ALP activity assay was conducted 3 days after the treatment. (B) Convergence of Tob and estrogen signaling at the transcriptional events. A luciferase reporter gene containing concatameric BMP response elements (GCCGx12) as well as Smad1, estrogen receptor, or Tob expression vectors were cotransfected into MC3T3E1 osteoblastic cells, which were then exposed to BMP2 and estrogen. Luciferase assay was conducted 3 days after the treatment.