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Clinical Trial
. 2012;7(3):e33168.
doi: 10.1371/journal.pone.0033168. Epub 2012 Mar 19.

Ablation of the pro-apoptotic protein Bax protects mice from glucocorticoid-induced bone growth impairment

Farasat Zaman  1 , Dionisios Chrysis, Kirsten Huntjens, Bengt Fadeel, Lars Sävendahl
Affiliations
Clinical Trial

Ablation of the pro-apoptotic protein Bax protects mice from glucocorticoid-induced bone growth impairment

Farasat Zaman et al. PLoS One. 2012.

Abstract

Dexamethasone (Dexa) is a widely used glucocorticoid to treat inflammatory diseases; however, a multitude of undesired effects have been reported to arise from this treatment including osteoporosis, obesity, and in children decreased longitudinal bone growth. We and others have previously shown that glucocorticoids induce apoptosis in growth plate chondrocytes. Here, we hypothesized that Bax, a pro-apoptotic member of the Bcl-2 family, plays a key role in Dexa-induced chondrocyte apoptosis and bone growth impairment. Indeed, experiments in the human HCS-2/8 chondrocytic cell line demonstrated that silencing of Bax expression using small-interfering (si) RNA efficiently blocked Dexa-induced apoptosis. Furthermore, ablation of Bax in female mice protected against Dexa-induced bone growth impairment. Finally, Bax activation by Dexa was confirmed in human growth plate cartilage specimens cultured ex vivo. Our findings could therefore open the door for new therapeutic approaches to prevent glucocorticoid-induced bone growth impairment through specific targeting of Bax.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Bax deficiency protects mice from Dexa-induced bone growth impairment.
Bax knockout (BaxKO) and wild-type female and male mice were treated with Dexa (2 mg/kg body weight/day) or saline for 28 days. X-ray images were captured on days 0, 7, 14, 21, and 28, and left femur lengths were measured. Femur bone length increase from day 0 in (A) female mice (***p<0.001, wild type Dexa vs. wild type vehicle; **p<0.01, wild type Dexa vs. BaxKO Dexa; n = 5) and (B) male mice (***p<0.001, wild type Dexa vs. wild type vehicle., n.s = not significant; n = 5.). Femur growth velocity (mm/day) in (C) female mice (***p<0.001, wild type Dexa vs. wild type vehicle.,**wild type Dexa vs. BaxKO Dexa., n = 5) and (D) male mice (***p<0.001, *p<0.05, wild type Dexa vs. wild type vehicle; n = 5). (E) TUNEL assays for the detection of apoptotic chondrocytes in growth plates of BaxKO and wild-type female mice treated with Dexa or vehicle for 28 days (**p<0.01, wild type Dexa vs. wild type vehicle., **p<0.01, wild type Dexa vs. BaxKO Dexa, n = 5). (F) BrdU incorporation analysis for the assessment of chondrocyte proliferation in growth plate cartilage of BaxKO and wild-type female mice treated with Dexa or saline for 28 days. (***p<0.001, wild type Dexa vs. wild type vehicle; **p<0.01, wild type Dexa vs. BaxKO Dexa; n = 5).
Figure 2
Figure 2. Loss of matrix and growth plate height.
(A) Tibial growth plate sections from female mice were stained with Alcian Blue to detect any alterations in matrix content within the growth plate cartilage. In wild-type mice, Alcian Blue staining appeared to be weaker in Dexa-treated animals compared to those treated with vehicle, while in BaxKO no such effect was seen. Furthermore, in Dexa-treated wild-type animals growth plate height appeared to be reduced and chondrocyte columns disorganized. To quantify any changes in matrix content, (B) loss of GAG was measured in the serum demonstrating significantly increased GAG release in Dexa treated wild type mice (*p<0.05, vs. wild type vehicle., n = 5). In contrast, BaxKO animals were protected from Dex-induced release of GAG (**p<0.01, vs. wild type Dexa; n = 5) (C) Representative micrographs of rat growth plate (tibia) showing different zones affected by Dexa; resting+proliferative (R+P) and hypertrophic (H) zones (bars represent 50 µm). Quantitative histological analysis of (D) growth plate height and (E) chondrocyte column density (columns per mm growth plate width) in vehicle- and Dexa-treated rats (**p<0.01, ***p<0.001; n = 6).
Figure 3
Figure 3. Bax ablation rescues chondrocytes.
Proliferative HCS chondrocytic cells treated with Dexa (25 µM) for 72 hrs were analyzed for the expression of the (A) Bax and (B) Bcl-2 proteins. (C) Bax ablation protected proliferative chondrocytes from Dexa-induced apoptosis. HCS-2/8 chondrocytic cells in proliferative phase were transfected with Bax-specific siRNAs (80 pmol) for 48 hrs prior to incubation with Dexa (25 µmol/L) for an additional 72 hrs. Suppression of Bax completely rescued chondrocytes from Dexa-induced apoptosis. Apoptosis was measured by using the Cell Death Detection ELISAPLUS kit. (D) Western immunoblotting showing suppression of Bax in Dexa-treated HCS-2/8 chondrocytic cells transfected with Bax-siRNA.
Figure 4
Figure 4. Conformational changes in Bax and translocation to mitochondria.
(A) Immunocytochemistry of HCS cells in proliferation phase, treated or untreated with Dexa (25 µM) for 72 hrs. Cells were analyzed for conformational changes in Bax using a specific antibody that only detects conformationally altered Bax. Chondrocytes were labeled with Bax antibody (red) and MitoTracker (green). Bax was mainly found in the mitochondria of Dexa-treated cells, as shown by the yellow-orange staining that is due to the merged red and green fluorescence. (B) Cultured fetal rat metatarsal bones treated with Dexa stained for DAPI (blue) and conformationally altered Bax (Conf- Bax; red). (C) Growth plate sections (tibia) of 7-week-old rats treated with Dexa (5 mg/kg/day) for 7 days. Immunohistochemistry was performed by triple-fluorescent labeling for conformationally changed Bax (Conf- Bax; red), HSP60 antibody (green), and DAPI (nuclei; blue). Conformationally changed Bax was mainly found in the chondrocyte mitochondria of those animals treated with Dexa, as shown by the yellow-orange staining that is due to the merging of red and green fluorescence.
Figure 5
Figure 5. Dexa triggers dissipation of mitochondrial membrane potential and cytochrome c release in human proliferative chondrocytes.
(A) Proliferative HCS-2/8 chondrocytic cells were treated with Dexa (25 µM) for 12, 24 and 48 hrs. Mitochondrial membrane potential was significantly decreased after 24 and 48 hrs in those chondrocytes treated with Dexa (***p<0.001 vs. untreated control). (B) HCS cells (treated or untreated with Dexa) were fractionated into cytosolic and mitochondrial extracts, and cytochrome c levels were determined in the cytosolic fractions. Cytochrome c was significantly increased following treatment with Dexa (25 µM) when assessed after 24, 48 and 72 hrs (***p<0.001 vs. untreated control). IGF-I (100 ng/ml) prevented Dexa-induced cytochrome c release at 24 hrs (**p<0.01 vs. Dexa alone), but failed to do so after 48 and 72 hrs. (C) Immunohistochemistry of cytochrome c release from mitochondria of HCS cells that were treated for 72 hrs with 25 µM Dexa or vehicle (99% EtOH). Cells were stained to visualize mitochondria (MitoTracker; green), cytochrome c (red), and nuclei (DAPI; blue). Cytochrome c was mainly found to be localized in the mitochondria (merged image-yellow) of control cells, while cytochrome c was evident in the cytosol of Dexa-treated HCS cells (shown as red fluorescence (arrows)). (D) Cytochrome c release was completely blocked by Bax siRNAs in transfected HCS-2/8 cells (**p<0.01; n = 4).
Figure 6
Figure 6. Activation of Bax in human growth plate cartilage.
Human growth plate biopsies from children undergoing epiphyseal surgery to reduce their longitudinal bone growth were treated with Dexa (1 µmol/L) and vehicle (99% EtOH) for 24 hrs. (A) Immunohistochemistry for the detection of conformationally altered active Bax was performed using a specific anti-Bax antibody (clone 6A7). (B) Quantification of percent cells staining positive for conformationally altered Bax (*p<0.05, n = 4).
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