Connexin 43 is required for the anti-apoptotic effect of bisphosphonates on osteocytes and osteoblasts in vivo

Abstract

Connexin (Cx)43 is required for inhibition of osteocyte and osteoblast apoptosis by bisphosphonates in vitro. Herein, we evaluated its requirement for the in vivo actions of bisphosphonates using mice in which Cx43 was deleted specifically from osteocytes and osteoblasts (Cx43(DeltaOb-Ot/-) mice). Effective removal of Cx43 was confirmed by the presence of the deleted form of the gene and by reduced mRNA and protein expression in osteoblastic cells and bones obtained from Cx43(DeltaOb-Ot/-) mice. The amino-bisphosphonate alendronate (2.3 micromol/kg/d) was injected daily into 5-mo-old female mice (n = 6-11) for 31 days, starting 3 days before implantation of pellets releasing the glucocorticoid prednisolone (2.1 mg/kg/d). Cx43(DeltaOb-Ot/-) mice and their littermates (Cx43(fl/-), Cx43(DeltaOb-Ot/+), and Cx43(fl/+)) gained bone with similar kinetics and exhibited identical bone mass from 2 to 4.5 mo of age, indicating that Cx43 deletion from osteocytes and mature osteoblasts does not impair bone acquisition. In addition, prednisolone induced a similar increase in osteocyte and osteoblast apoptosis in Cx43(DeltaOb-Ot/-) or in control Cx43(fl/-) littermates. However, whereas alendronate prevented prednisolone-induced apoptosis in control Cx43(fl/-) mice, it was ineffective in Cx43(DeltaOb-Ot/-) mice. In contrast, alendronate inhibited glucocorticoid-induced bone loss in both type of animals, suggesting that inhibition of resorption is the predominant effect of alendronate against the early phase of glucocorticoid-induced bone loss. Taken together with earlier in vitro evidence, these findings show that Cx43 is required for the anti-apoptotic effect of bisphosphonates on osteocytes and osteoblasts.

FIG. 1

Genotypic and phenotypic analysis of mice lacking Cx43 in osteocytes and osteoblasts and their littermates. (A) Genomic DNA was purified from mouse bone (tibia) and heart and PCR for OCNCre, “floxed,” and wildtype Cx43 allele and deleted Cx43 were performed. (B) Calvaria cells were isolated from mice carrying the four different genotypes and treated with ascorbic acid for 0 or 21 days. The levels of Cx43 mRNA were determined by real-time PCR and corrected by ChoB. The expected levels of expression of Cx43 are indicated. *p < 0.001 vs. day 0, n = 3–9 mice. (C) Representative microphotographs of paraffin-embedded bone (tibia) and heart sections immunostained for Cx43 (brown) and counterstained with methyl green to show the cell nuclei (blue nuclei). An example of an osteocyte in each bone section is pointed out by black arrows and an example of an osteoblast by white arrows.

FIG. 2

Cx43^{ΔOb−Ot/−} mice that lack Cx43 in osteocytes/osteoblasts do not differ in the rate of gain of weight or global, spinal, or hindlimb BMD from their littermates. Weight and global, spinal, and hindlimb BMD were determined every 2 wk starting at 2 mo of age until the mice reached peak bone mass (4.3 mo old). Six to 12 mice were analyzed for each genotype. p > 0.05, by repeated measures models for all measurements for either female or male mice indicate the lack of differences among the groups.

FIG. 3

Alendronate prevents glucocorticoid-induced cancellous and cortical osteocyte and osteoblast apoptosis in Cx43^fl/− but not in Cx43^{ΔOb−Ot/−} mice lacking Cx43 in osteocytes and osteoblasts. Mice were treated with daily alendronate injections, starting 3 days before pellet implantation. Twenty-eight days after pellet implantation, mice were killed, and apoptosis was determined. pred, prednisolone; n.s., not significant. ^a p < 0.05 by generalized linear models, n = 4–9 mice.

FIG. 4

Alendronate prevents glucocorticoid-induced bone loss in both Cx43^fl/− and Cx43^ΔOb-Ot/− mice. Mice were treated as indicated in Fig. 3. Initial BMD was determined when the pellets were implanted and the final BMD at the time of death. The percent change in BMD was calculated. pred, prednisolone. ^a p < 0.05 by generalized linear models, n = 6–11 mice.

FIG. 5

Alendronate does not prevent apoptosis of cultured osteocytic and osteoblastic cells silenced for Cx43. The expression of Cx43 was silenced in MLO-Y4 osteocytic cells (A) and Ob-6 osteoblastic cells (B) using short hairpin RNA (shRNA)-containing virus. As controls, cells were infected with scrambled (scr) shRNA. Cx43 protein levels were determined by Western blotting and Cx43 mRNA levels by real time RT-PCR. Cells were cultured for 1 h with vehicle or 10⁻⁷ M alendronate, followed by 6-h treatment with vehicle or 10⁻⁶ M dexamethasone. Dead cells were enumerated by trypan blue uptake. dex, dexamethasone; n.s., not significant. ^a p < 0.05 by one-way ANOVA.