Cytosolic interaction between AIF and Scythe is required for macrophage clearance of Fas-triggered Jurkat cells.

<p>A. Immunocytochemistry analysis of Scythe and AIF in Jurkat cells un-treated and treated for 3 h with Fas mAb (250 ng/ml) in the presence or absence of the pan-caspase inhibitor, Z-VAD-FMK (20 µM). Nuclei are stained in blue (DAPI). B. Jurkat cells treated like in A were lysed and AIF was then immunoprecipitated. Western blot is 10% of the input and is derived from both control and Fas mAb treated cells. Negative control (NC) consists of glass beads incubated with normal rabbit serum. C. Jurkat cells were transfected with siRNA for AIF, Scythe and non-targeting siRNA (NC). After 48 h cells were collected, lysed in RIPA buffer and Western blotting analysis was performed to detect the levels of AIF. GAPDH was used as loading control. Quantification was made using the program Image J and the values of optical density (OD) were normalized to the levels of GAPDH. D. Jurkat cells, treated with siRNA for AIF, Scythe or NC were induced to undergo apoptosis by Fas mAb (50 ng/ml) or staurosporine (0.5 µM). After 3 h cells were co-cultivated with human monocyte-derived macrophages (HMDM) for 1 h. Examples of phagocytosis of TAMRA-labelled Jurkat cells (red) are depicted. Nuclei of macrophages and engulfed target cells are stained with DAPI (blue). E. Phagocytosis is reported as the percentages of macrophages positive for uptake of target cells. Data are presented as the mean ± S.D of six values derived from two different experiments performed in triplicate. *P<0.05; **P<0.01 (one-way ANOVA <i>Dunnett’s</i> test). Caspase-3 activation in WT versus AIF or Scythe siRNA-transfected cells determined by immunoblotting is shown in the panel below. GAPDH is used as loading control.</p

AIF and Scythe (Bat3) Regulate Phosphatidylserine Exposure and Macrophage Clearance of Cells Undergoing Fas (APO-1)-Mediated Apoptosis

<div><p>Phosphatidylserine (PS) exposure on the cell surface has been considered a characteristic feature of apoptosis and serves as a molecular cue for engulfment of dying cells by phagocytes. However, the mechanism of PS exposure is still not fully elucidated. Here we show that the cytosolic release from mitochondria of apoptosis-inducing factor (AIF) is required for PS exposure during death receptor-induced apoptosis and for efficient clearance of cell corpses by primary human macrophages. Fas-triggered PS exposure was significantly reduced upon siRNA-mediated silencing of AIF expression and by inhibition of the cytosolic translocation of AIF. In addition, AIF localizes to the plasma membrane upon Fas ligation and promotes activation of phospholipid scrambling activity. Finally, cytosolic stabilization of AIF through interaction with Scythe is shown to be involved in apoptotic PS exposure. Taken together, our results suggest an essential role for AIF and its binding partner Scythe in the pathway leading to apoptotic corpse clearance.</p> </div

AIF is required for the activation of the phospholipid scramblase activity.

<p>A. Jurkat cells WT and knockdown for AIF (48 h) were stained with the lipid raft marker, cholera toxin B (CTB) and with an anti-AIF antibody and subjected to immunofluorescence imaging. Co-localization between AIF and lipid rafts was validated by image analysis using the program Image J. B. Fluorescent NBD-PS was used to monitor scramblase activity in apoptotic cells. Following transfection of Jurkat cells with siRNA as indicated (48 h), cells were incubated with NBD-PS (10 µM) for 10 min at 28°C. Then, cells were treated with 50 ng/ml Fas mAb for 3 h, after which the percentage of residual fluorescence (due to the reduction of the NBD-PS probe) was determined using a Fluoroscan II plate reader with an excitation wavelength of 470 nm and emission wavelength = 540 nm. Data are presented as the mean± S.D of six values derived from two different experiments performed in triplicate. *P<0.05 (one-way ANOVA <i>Dunnett’s</i> test).</p

Inhibition of AIF cytosolic release reduces PS exposure in Fas-treated Jurkat cells.

<p>A. Jurkat cells were treated with Fas mAb (50 ng/ml) with or without pre-treatment for 2 h with bongkrekic acid (BA) (50 µg/ml). Cells were stained with the mitochondrial marker, MitoTracker Red. Co-localization of AIF and mitochondria was validated by image analysis using the program Image J. B. Jurkat cells treated as above were collected after 3 h and Annexin V-FITC staining was performed to monitor the level of PS exposure. C. Jurkat cells treated as above were lysed and real-time in vitro assay for caspase-3 was performed using the fluorogenic substrate DEVD-AMC. Results are reported as pmol/min of substrate cleavage. Data are presented as the mean± S.D of nine values derived from three different experiments performed in triplicate (one-way ANOVA <i>Dunnett’s</i> test).</p

Down-regulation of AIF reduces PS exposure in Jurkat cells without affecting caspases activation.

<p>A. Jurkat cells were transfected with siRNA for AIF and non-targeting siRNA (NC) in 12-well culture plates. After 48 h cells were collected, lysed in RIPA buffer and Western blotting analysis was performed to detect the levels of AIF. GAPDH was used as loading control. Quantification was made using the program Image J and the values of optical density (OD) were normalized according to the levels of GAPDH. B. Jurkat cells transfected like in A were treated with Fas mAb (50 ng/ml) or staurosporine (0.5 µM) and after 3 h were lysed and enzymatic assay for caspase-3 was performed using the fluorogenic substrate DEVD-AMC. Results are reported as fold increase compared to control. Data are presented as the mean± S.D of nine values derived from three different experiments performed in triplicate (one-way ANOVA <i>Dunnett’s</i> test). C. Jurkat cells transfected like in A were treated with Fas mAb (50 ng/ml) or staurosporine (0.5 µM). After 3 h, cells were collected and lysed and Western blotting was performed to detect active caspase-3 and PARP cleavage. GAPDH was used as loading control. D. Jurkat cells transfected like in A were treated with Fas mAb (50 ng/ml) or staurosporine (0.5 µM). After 3 h, cells were collected and Annexin V-FITC staining was performed to determine the level of PS exposure.</p

Conformational changes in Bax and translocation to mitochondria.

<p>(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.</p

Loss of matrix and growth plate height.

<p>(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., <i>n</i> = 5). In contrast, BaxKO animals were protected from Dex-induced release of GAG (**p<0.01, <i>vs.</i> wild type Dexa; <i>n</i> = 5) (C) Representative micrographs of rat growth plate (tibia) showing different zones affected by Dexa; resting+proliferative (<i>R</i>+<i>P</i>) and hypertrophic (<i>H</i>) zones (<i>bars</i> 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; <i>n</i> = 6).</p

Dexa triggers dissipation of mitochondrial membrane potential and cytochrome c release in human proliferative chondrocytes.

<p>(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 <i>vs.</i> 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 <i>vs.</i> untreated control). IGF-I (100 ng/ml) prevented Dexa-induced cytochrome c release at 24 hrs (**p<0.01 <i>vs.</i> 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; <i>green</i>), cytochrome c (<i>red</i>), and nuclei (DAPI; <i>blue</i>). Cytochrome c was mainly found to be localized in the mitochondria (merged image-<i>yellow</i>) of control cells, while cytochrome c was evident in the cytosol of Dexa-treated HCS cells (shown as red fluorescence (<i>arrows</i>)). (D) Cytochrome c release was completely blocked by Bax siRNAs in transfected HCS-2/8 cells (**p<0.01; <i>n = 4</i>).</p

Activation of Bax in human growth plate cartilage.

<p>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, <i>n</i> = 4).</p

Bax deficiency protects mice from Dexa-induced bone growth impairment.

<p>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; <i>n</i> = 5) and (B) male mice (***p<0.001, wild type Dexa vs. wild type vehicle., n.s = not significant; <i>n</i> = 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., <i>n</i> = 5) and (D) male mice (***p<0.001, *p<0.05, wild type Dexa vs. wild type vehicle; <i>n</i> = 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, <i>n</i> = 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; <i>n</i> = 5).</p