Sphingolipid metabolism cooperates with BAK and BAX to promote the mitochondrial pathway of apoptosis

Abstract

Mitochondria are functionally and physically associated with heterotypic membranes, yet little is known about how these interactions impact mitochondrial outer-membrane permeabilization (MOMP) and apoptosis. We observed that dissociation of heterotypic membranes from mitochondria inhibited BAK/BAX-dependent cytochrome c (cyto c) release. Biochemical purification of neutral sphingomyelinases that correlated with MOMP sensitization suggested that sphingolipid metabolism coordinates BAK/BAX activation. Using purified lipids and enzymes, sensitivity to MOMP was achieved by in vitro reconstitution of the sphingolipid metabolic pathway. Sphingolipid metabolism inhibitors blocked MOMP from heavy membrane preparations but failed to influence MOMP in the presence of sphingolipid-reconstituted, purified mitochondria. Furthermore, the sphingolipid products, sphingosine-1-PO(4) and hexadecenal, cooperated specifically with BAK and BAX, respectively. Sphingolipid metabolism was also required for cellular responses to apoptosis. Our studies suggest that BAK/BAX activation and apoptosis are coordinated through BH3-only proteins and a specific lipid milieu that is maintained by heterotypic membrane-mitochondrial interactions.

Figure 1. Microsomes Restore Mitochondrial Sensitivity to Direct Activator Stimulation

(A) HM and mitochondria were treated with C8-BID (0.5, 1, and 5 nM). (B) The HM fraction was isolated, and the μS and mitochondrial (Mito) fractions were purified. All three fractions were subjected to western blot. In parallel, each fraction was incubated with 10 nM C8-BID for 1 hr at 37°C, washed, and subjected to western blot. (C) Same as in (A), but treatments included BID BH3 and BIM BH3 (1, 5, and 20 μM). (D) HM (50 μg) and mitochondria (50 μg) were treated as indicated (C8-BID, 1 nM; BID BH3, 5 μM; BCL-xL, 500 nM; μS, 5, 25, and 50 μg). (E) HM, mitochondrial, and μS fractions were subjected to western blot. (F) HM, mitochondrial, and μS fractions were treated with limited proteolysis, lysed, and subjected to western blot. (G) Purification scheme to identify SMPD3 and SMPD4. See also Figure S1.

Figure 2. A Neutral SMase Activity Cooperates within the BCL-2 Family to Promote MOMP

(A) SMPD2-4 IVTT with ³⁵S-Met were subjected to SDS-PAGE. Lines denoting 2, 3, and 4 indicate full-length products. (B) Nonradioactive IVTT reactions were performed and subjected to ¹⁴C-SM hydrolysis ± 10 μM GW4869. (C) Nonradioactive IVTT reactions were performed and combined with WT HM fractions. C8-BID (1 nM for control; 0.05 nM for IVTT combinations); GW4869 (25 μM) and BCL-xL (50 nM). (D) WT HM fractions were pretreated with FB1 (1, 5, 10, and 25 μM), bSMase (0.0001, 0.0005, 0.0010, and 0.0025U) or D609 (1, 5, and 25 μM) for 1 hr at 37°C before the addition of 0.05 nM C8-BID. (E) Liver homogenate, solubilized HM fraction, or solubilized mitochondria (1 μg) were subjected to ¹⁴C-SM hydrolysis. (F) HM fraction was subjected to limited proteolysis (0, 1, 2, 5, 10, and 20 min) on ice, and fractionated into pellet (mitochondria) and supernatant (contains released μS) before ¹⁴C-SM hydrolysis and western blot. (G) WT HM fractions were combined with C8-BID (0.05, 0.1, 0.5, and 1 nM) or as indicated in the presence of SMPD3 (1, 5, and 20 ng) and BCL-xL (500 nM). (H) bak^-/-bax^-/- HM fractions treated with SMPD3 (20 ng; or 1, 20 ng), BAX (25 nM), C8-BID (10 nM; or 1, 10 nM; or as indicated), and BCL-xL (500 nM). (I) SMPD3 was tested for ¹⁴C-SM hydrolysis and compared to bSMase. All data are reported as ± SD. See also Figure S2.

Figure 3. Inhibition of the Sphingolipid Pathway Blocks MOMP

(A) Substrates, products, and enzymes, together with enzyme inhibitors tested. (B–D) WT HM fractions were pretreated with the indicated drugs for 1 hr at 37°C prior to addition of C8-BID (1 nM). GW4869 (2, 10, and 20 μM), MAPP (2, 10, and 20 μM), NOE (0.1, 0.5, and 1 μM), DMS (2.5, 12.5, and 25 μM), SKI (5, 25, and 50 μM), DOP (1, 5, and 10 μM), THI (1, 5, and 10 μM). (E) HM and subsequent mitochondria were purified in the presence of DOP/THI (10 μM each) before treatment with C8-BID (0, 0.5, and 1 nM). (F) The levels of S1P were measured following isolation as in (E). (G and H) bak^-/-bax^-/- HM fractions were pretreated with drugs (two higher doses from above) for 1 hr at 37°C prior to addition of BAX (25 nM) and C8-BID (10 nM). (I) The HM fraction was pretreated with the highest dose of the inhibitors listed in (B)–(D) for 1 hr at 37°C, prior to addition of BAX (25 nM) and C8-BID (10 nM). Reactions were then incubated for 1 hr at 37°C before lysis in 1% CHAPS buffer and IP. A MOMP reaction lysed in 1% Triton X-100 is the positive control. All graphed data are reported as ± SD. See also Figure S3.

Figure 4. Reconstitution of the Sphingolipid Pathway Replaces the Heterotypic Membrane Requirement for MOMP

(A) Purified mitochondria were preincubated for 1 hr with indicated sphingolipids (SM or C16, 1 μM), purified sphingolipid enzymes (bSMase or ASAH2, 0.001 U/μl) or drugs (GW4869 or MAPP, 25 μM) before the addition of C8-BID (1 nM). SPH was added at 0.1 or 1 μM. (B) WT mitochondria were incubated with indicated sphingolipids (0.1, 0.5, and 1 μM) prior to the addition of C8-BID (1 nM). As a positive control, purified μS (50 μg) was also tested for sensitization. (C) bak^-/-bax^-/- mitochondria were treated with indicated sphingolipids (1 μM). (D) bak^-/-bax^-/- HM fractions and mitochondria were pretreated with BAX (25 nM), C8-BID (10 nM), and indicated sphingolipids (0.1 and 1 μM). (E) BAX was incubated with hex (0.1, 0.5, and 1 μM) and C8-BID (5, 10, and 25 nM) for 1 hr at 37°C before IP with anti-BAX 6A7. Triton X-100 (0.5%) was added as a positive control for detergent-treatment activation. (F) LUVs (PC, PE, PI, PS, no CL) were treated with indicated proteins and hex. (G) Mitochondria were pretreated with CsA (100 nM) for 1 hr at 37°C prior to the addition of S1P (0.1, 0.5, and 1 μM) and C8-BID (1 nM). All data are reported as ± SD. See also Figure S4.

Figure 5. Hexadecenal Binds and Promotes BAX Activation

(A) BAX (10 nM) was incubated with indicated lipids or detergents (0.1 and 1 mM) in the presence of BMH (100 nM) for 1 hr at 37°C before western blot. (B) As in (A), but instead of oligomerization analysis, samples were extensively dialyzed, combined with bak^-/-bax^-/- HM, and incubated for 1 hr at 37°C before fractionation, and western blot for cyto c. (C and D) Nitrocellulose membranes spotted with indicated picomoles of S1P or hex (combined with BSA and ponceau red for visualization) were incubated with either 50 ng of BCL-xL or BAX and probed with anti-BCL-xL or anti-BAX (horizontal mark for anti-BCL-xL, vertical for anti-BAX). The positive controls (*) on the blots are spots containing an IP antibody for BCL-xL or BAX. (E) BAX (10 nM) was incubated with hexadecanal or hex (0, 0.1, 1, 5, and 10 μM) in the presence of BMH (100 nM) for 1 hr at 37°C. See also Figure S5.

Figure 6. SMPD3 and SMPD4 Regulate Sensitivity to Apoptosis, and a Mitochondrial Sphingosine Kinase Activity Sensitizes for MOMP

(A) HeLa cells were transfected with vector, SMPD3, or SMPD4; or treated with CHX (10 μg/ml), TNF (25 ng/ml; or 5, 10, 15, 20, and 25 ng/ml); cultured for 24 hr and stained with Annexin V-PE. (B) HeLa cells were transfected with 100, 500, 1000 ng of vector, SMPD3 or SMPD4, cultured for 24 hr, treated with CHX (10 μg/ml) and TNF (15 ng/ml), cultured for 6 hr and stained with Annexin V-PE. (C) WT MEFs retrovirally infected to silence sphk1 or sphk2 were analyzed by western blot. (D) Control, sphk1, and sphk2 RNAi cell lines were treated with CHX (5 μg/ml) and TNF (10 ng/ml), cultured for 6 hr, and stained with Annexin V-PE. (E) HM fraction, mitochondria, and μS were subjected to western blot. (F) WT HM fractions were treated with C8-BID (5, 25, 1000* pM, *positive control), SPHK1 (25 ng), SPHK2 (25 ng), BCL-xL (50 nM) in MAB or RB. See also Figure S6.

Figure 7. The SMase Pathway Regulates Sensitivity to Apoptosis

(A) HeLa cells were pretreated with indicated pharmacological inhibitors for 6 hr, apoptotic inducers (CH11/CHX, 0.4 μg/ml + 10 μg/ml CHX; 30 ng/ml TNF + 10 μg/ml CHX; or 50 nM ActD) added for 24 hr, and colonies were stained with methylene blue 12 days later. S1P (0.25 μM) was added at the same time as the inhibitors. (B–D) Colonies from CH11/CHX, TNF/CHX, and ActD treatments are shown. Wells were treated as indicated (top to bottom, left to right): (1) DMSO, (2) 50 μM D609, (3) 20 μM GW4869, (4) 100 μM DOP, (5) 100 μM THI, and (6) 0.25 μM S1P. All data are reported as ± SD. See also Figure S7.