Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin

Abstract

Glucocorticoid-induced osteoporosis may be due, in part, to increased apoptosis of osteocytes and osteoblasts, and bisphosphonates (BPs) are effective in the management of this condition. We have tested the hypothesis that BPs suppress apoptosis in these cell types. Etidronate, alendronate, pamidronate, olpadronate, or amino-olpadronate (IG9402, a bisphosphonate that lacks antiresorptive activity) at 10(-9) to 10(-6) M prevented apoptosis of murine osteocytic MLO-Y4 cells, whether it was induced by etoposide, TNF-alpha, or the synthetic glucocorticoid dexamethasone. BPs also inhibited apoptosis of primary murine osteoblastic cells isolated from calvaria. Similar antiapoptotic effects on MLO-Y4 and osteoblastic cells were seen with nanomolar concentrations of the peptide hormone calcitonin. The antiapoptotic effect of BPs and calcitonin was associated with a rapid increase in the phosphorylated fraction of extracellular signal regulated kinases (ERKs) and was blocked by specific inhibitors of ERK activation. Consistent with these in vitro results, alendronate abolished the increased prevalence of apoptosis in vertebral cancellous bone osteocytes and osteoblasts that follows prednisolone administration to mice. These results suggest that the therapeutic efficacy of BPs or calcitonin in diseases such as glucocorticoid-induced osteoporosis may be due, in part, to their ability to prevent osteocyte and osteoblast apoptosis.

Figure 1

Demonstration of MLO-Y4 cell apoptosis. (a) MLO-Y4 cells were incubated for 1 hour in vehicle or 50 nM DEVD-CHO. Subsequently, etoposide, TNF-α, or dexamethasone (Dex) were added at final concentrations of 50 μM, 1 nM, or 10^–6 M, respectively, and cells were incubated for an additional 6 hours. Dead cells were enumerated by trypan blue uptake, as described in Methods. Bars represent the mean ± SD of 3 independent measurements. Data were analyzed by 1-way ANOVA. *P < 0.05 versus control (Student Newman-Keuls method). (b) MLO-Y4 cells stably transduced with GFP were maintained for 6 hours in the presence of vehicle, 50 μM etoposide, 1 nM TNF-α, or 10^–6 M dexamethasone. Numbers indicate the percentage of cells undergoing apoptosis, as determined by evaluating the nuclear morphology of more than 500 cells in fields selected by systematic random sampling. Results are means ± SD of 3 independent experiments. Original magnification: ×400. Data were analyzed by 1-way ANOVA. *P < 0.05 versus control (Student Newman-Keuls method).

Figure 2

Bisphosphonates inhibit glucocorticoid-induced apoptosis of MLO-Y4 osteocytic cells. MLO-Y4 cells were pretreated with the indicated concentrations of BPs for 1 hour, and subsequently dexamethasone (10^–6 M final concentration) was added for 6 hours. The percentage of dead cells was determined by trypan blue uptake, as in Figure 1a. Bars represent the mean ± SD of 3 independent measurements. Data were analyzed by 1-way ANOVA. *P < 0.05 versus dexamethasone alone (Student Newman-Keuls method).

Figure 3

Bisphosphonates inhibit glucocorticoid-induced apoptosis of MLO-Y4 osteocytic cells. MLO-Y4 cells stably transduced with GFP were treated for 1 hour with 10^–7 M concentration of the indicated bisphosphonates, followed by addition of 10^–6 M dexamethasone. After 6 hours, cells were fixed. Numbers indicate the percentage of cells undergoing apoptosis as determined by evaluating the nuclear morphology of more than 500 cells in fields selected by systematic random in at least 3 different experiments (mean ± SD). Original magnification: ×400. Data were analyzed by 1-way ANOVA. *P < 0.05 versus control (Student Newman-Keuls method). The effect of bisphosphonates on the proportion of MLO-Y4 cells exhibiting chromatin condensation and/or nuclear fragmentation in the absence of dexamethasone was also analyzed using exact χ² tests in 3 experiments with pamidronate, olpadronate, or IG9402, and in 6 experiments with alendronate. Bonferroni adjusted pairwise comparisons between untreated groups and groups treated with bisphosphonates yielded no significant differences when the experiments were analyzed individually; however, differences were significant when all experiments for given treatment condition were combined in a stratified χ² analysis (see the text).

Figure 4

Bisphosphonates inhibit the increase in caspase-3 activity and the decrease in the number of living cells induced by dexamethasone. MLO-Y4 cells were incubated with vehicle or 10^–7 M alendronate for 1 hour. Subsequently, dexamethasone was added to reach a final concentration of 10^–6 M, and cells were cultured for 16 hours (a) or 24 hours (b). Caspase-3 activity was determined by measuring the degradation of the fluorometric substrate DEVD-AFC in the absence or presence of the irreversible inhibitor DEVD-CHO, as detailed in Methods (a). Cells were harvested by trypsinization, and the number of living cells was scored using an hemocytometer as described in Methods (b). Bars represent the mean ± SD of 3 independent measurements. *P < 0.05 versus control, by ANOVA (Student Newman-Keuls method).

Figure 5

The protective effect of bisphosphonates on osteocytic cells is independent of the proapoptotic stimulus. MLO-Y4 cells were incubated with vehicle, 10^–7 M alendronate, or IG9402 for 1 hour. Subsequently, etoposide, TNF-α, or dexamethasone were added to reach final concentrations of 50 μM, 1 nM, or 10^–6 M, respectively, and cells were cultured for 6 hours, as in Figure 1. The percentage of dead cells was determined by trypan blue uptake. Bars represent the mean ± SD of 3 independent measurements. Data were analyzed by 2-way ANOVA. No interaction between the proapoptotic agents and the pretreatments was found (at an α level of 0.05). *P < 0.05 versus etoposide, TNF-α, or dexamethasone alone (Student Newman-Keuls method).

Figure 6

The protective effect of bisphosphonates on osteoblastic cells is independent of the proapoptotic stimulus. This experiment was performed as in Figure 5, but using osteoblastic cells derived from murine calvaria. Bars represent the mean ± SD of 3 independent measurements. Data were analyzed by 2-way ANOVA. No interaction between the proapoptotic agents and the pretreatments was found (at an α level of 0.05). *P < 0.05 versus etoposide, TNF-α, or dexamethasone alone (Student Newman-Keuls method).

Figure 7

Time course of ERK activation by alendronate and IG9402. (a) MLO-Y4 cells were stimulated with 10^–7 M alendronate (A) or IG9402 (IG) for the indicated times. Phosphorylated ERK1/2 and total ERK1/2 were determined by Western blot analysis as described in Methods. (b) Phospho-ERK/ERK ratios were obtained by quantifying the intensity of the bands in the autoradiograms using a scanner.

Figure 8

The antiapoptotic effect of BPs involves ERK activation. (a) Cells were incubated for 23 minutes with vehicle, 50 μM PD98059, or 1 μM UO126 before addition of 10^–7 M alendronate (A), IG9402 (IG), or etidronate (E) for 2 minutes. Cell lysates were obtained and assayed for the presence of phosphorylated ERK1/2 as in Figure 7. Cells were treated for 30 minutes with PD98059 (b) or with UO126 (c), followed by addition of 10^–7 M BPs. After 1 hour, 10^–6 M dexamethasone was added and cultures were incubated for 6 hours. The percentage of apoptotic cells was determined by trypan blue exclusion, as in Figure 1a. Bars represent the mean ± SD of 3 independent measurements. *P < 0.05 versus control by 1-way ANOVA (Student Newman-Keuls method).

Figure 9

Salmon calcitonin inhibits glucocorticoid-, TNF-α–, and etoposide-induced apoptosis of MLO-Y4 and osteoblastic cells. (a) MLO-Y4 or UMR-106-06 cells were incubated with 10^–8 M [¹²⁵I]sCT in the absence or in the presence of 10^–6 M unlabeled sCT for 1 hour at room temperature. After washing, bound [¹²⁵I]sCT was determined. (b) Concentration of cAMP in MLO-Y4 cells upon treatment with 5 ng/mL of sCT was measured using a commercially available kit, as described in Methods. Each point represents triplicate determinations ± SD. (c) MLO-Y4 osteocytic cells or osteoblastic cells were treated with sCT for 1 hour before the addition of the proapoptotic stimuli. The percentage of dead cells was determined by trypan blue uptake as described in Figure 1a. Bars represent the mean ± SD of 3 independent measurements. *P < 0.05 versus etoposide, TNF-α, or dexamethasone alone by 1-way ANOVA (Student Newman-Keuls method).

Figure 10

The antiapoptotic effect of salmon calcitonin involves ERK activation. (a) MLO-Y4 cells were stimulated with 5 ng/mL of sCT for the indicated times. Phosphorylated ERK1/2 and total ERK1/2 were determined by Western blot analysis as described in Methods. (b) Cells were treated for 30 minutes with PD98059 or with UO126, followed by addition of 5 ng/mL of sCT. After 1 hour, 10^–6 M dexamethasone was added and cultures were incubated for 6 hours. The percentage of apoptotic cells was determined by trypan blue exclusion, as in Figure 1a. Bars represent the mean ± SD of 3 independent measurements. *P < 0.05 versus control by 1-way ANOVA (Student Newman-Keuls method).

Figure 11

Effect of prednisolone on murine osteocyte and osteoblast apoptosis in vivo. Photomicrographs are from vertebral cancellous bone of mice treated for 56 days with prednisolone. Apoptotic, TUNEL-positive osteocytes (a) and osteoblasts (b) are indicated by the arrows. A normal osteocyte (a) and a normal osteoblast (b) are indicated by arrowheads. Methyl green counterstain viewed with Nomarski differential interference microscopy. Original magnification: ×1,000 and ×630, for a and b, respectively.

Figure 12

Functional syncytium comprising osteocytes, osteoblasts, bone marrow stromal cells, and endothelial cells. See the text for details. (Adapted with permission from G. Marotti [64].)