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Published as doi: 10.1096/fj.07-8967com.
 (The FASEB Journal. 2008;22:183-193.)
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(The FASEB Journal. 2008;22:183-193.)
© 2008 FASEB

A novel role for protein kinase C{delta}-mediated phosphorylation of acid sphingomyelinase in UV light-induced mitochondrial injury

Youssef H. Zeidan*, Bill X. Wu*, Russell W. Jenkins*, Lina M. Obeid{dagger} and Yusuf A. Hannun*,1

* Departments of Biochemistry and Molecular Biology and

{dagger} Medicine, Medical University of South Carolina, Charleston, South Carolina, USA

1Correspondence: Department of Biochemistry and Molecular Biology, Medical University of South Carolina,175 Ashley Ave., P.O. Box 250509, Charleston, SC 29425, USA. E-mail: hannun{at}musc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Multiple studies have addressed the mechanisms by which ultraviolet (UV) light induces cell death, and a few have focused on stress mediators such as acid sphingomyelinase (ASMase) or protein kinase C{delta} (PKC{delta}). Based on a recent study that identified a novel mechanism of activation of ASMase through phosphorylation (1) , the current study was undertaken to determine the upstream mechanisms regulating ASMase in response to UV and to investigate the role of ASMase and its phosphorylation at S508 as an integral event during UV light-induced cell death. Exposure of MCF-7 breast cancer cells to UV light type C (UVC) transiently activated ASMase with maximal activity detected at 10 min postirradiation. A significant increase in C16-ceramide was detected concomitant with a decrease in C16-sphingomyelin. In marked contrast, cells overexpressing the ASMaseS508A mutant, which could not be phosphorylated, had no change in either ASMase activity or ceramide levels post-UV radiation. Loss of PKC{delta} by RNA interference or its inhibition by rottlerin blocked ASMase phosphorylation and membrane targeting, thus implicating PKC{delta} upstream of ASMase activation by UV light. Further investigations revealed that UV radiation altered mitochondrial morphology from elongated tubules to fragmented perinuclear organelles, consistent with the onset of the apoptotic cascade. Importantly, cells overexpressing ASMaseS508A were protected (>50%) from UV light-induced mitochondrial fragmentation. Mechanistically, the results showed that ASMaseS508A cells had 50% less active Bax than ASMaseWT cells. These molecular differences culminated in resistance of ASMaseS508 cells to UVC-induced cell death (25%) as compared to ASMaseWT cells (46%). Taken together, this study provides key molecular insights into activation of ASMase in response to UV light, the role of PKC{delta} in this activation, and the role of ASMase in mediating apoptotic responses.—Zeidan, Y. H., Wu, B. X., Jenkins, R. W., Obeid, L. M., Hannun, Y. A. A novel role for protein kinase c{delta}-mediated phosphorylation of acid sphingomyelinase in UV light-induced mitochondrial injury.


Key Words: ceramide • sphingolipids • apoptosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ULTRAVIOLET (UV) RADIATION IS A ubiquitous environmental mutagen involved in sunlight-induced carcinogenesis (2) . As a protective mechanism, cells that fail to repair UV injury are normally targeted to undergo programmed cell death (apoptosis; ref. 3 ). Dysfunctions in these responses can lead to formation of tumors. Thus, understanding the molecular mechanisms underlying UV-induced apoptosis is critical.

Cumulative efforts from a number of laboratories established that induction of apoptosis by UV radiation involves elements of the intrinsic as well as the extrinsic death pathways (4) . Of major importance within the intrinsic pathway of UV-induced apoptosis are the tumor suppressors p53, p21, and Gadd 45 (5 6 7 8) . Alternatively, UV radiation not only increases the expression of the CD95/FasL system but also clusters CD95 within structured membrane domains (so called lipid rafts) through a JNK cell signaling pathway (9 10 11 12 13) . Despite remarkable progress, a gap still exists in the current understanding of the early signaling events triggered by UV exposure.

Previous studies have suggested a pivotal role for protein kinase C {delta} (PKC{delta}) in mediating the effects of UV light (14 , 15) , whereas other studies have focused on the role of sphingomyelinases (SMases) and the bioactive lipid ceramide as candidate mediators of cytotoxic effects of UV light (16 17 18 19 20) . Interestingly, in a recent study, we provided a mechanistic link between PKC{delta} and acidic sphingomyelinase (ASMase; ref. 1 ), and this prompted us to investigate the mechanisms of UV-induced cell death focusing on the role of PKC{delta} and ASMase.

Protein kinase C (PKC) comprises a family of 10 serine/threonine kinases integral to signal transduction pathways. Based on their structural homology and cofactor requirements, PKCs are classified to three families: classical ({alpha}, βI, βII, {gamma}), novel ({delta}, {epsilon}, {eta}, {theta}), and atypical ({zeta}, {iota}/{lambda}). Recent studies have established PKCs as important bioregulators of sensitivity vs. resistance of mammalian cells to UV-induced apoptosis (21 , 22) . While the atypical PKC{zeta} confers a resistant phenotype (23) , the novel PKC{delta} emerged as a pivotal effector of UV-induced apoptosis (24) . Studies by Denning and coworkers (14) demonstrated that UV light activates PKC{delta} by cleavage at the hinge region through a caspase-dependent mechanism. Overexpression of a noncleavable PKC{delta} mutant or addition of PKC{delta} pharmacologic inhibitors prevented the death of human keratinocytes (25) . Of note, cleavage of PKC{delta} in this case appears to be a late event in activation of apoptotic programs (downstream of caspases) and it seems to be cell line specific as it was not observed in other cell types such as HaCat (15) .

Importantly, activation of PKC{delta} by UV light has been reported to differentially regulate proapoptotic and antiapoptotic Bcl-2 proteins. In one study, it was demonstrated that the catalytic form of PKC{delta} mediates Bax up-regulation and activation by UV light (26) . In a follow up study, it was found that direct phosphorylation of Mcl-1 by PKC{delta} promotes its proteolytic degradation during the UV response (27) . Therefore, elucidation of the direct PKC{delta} targets is crucial to fully understand its contribution to the apoptotic cascade.

Ceramide has emerged at the forefront of sphingolipid biology as a pleiotropic bioactive lipid that regulates many responses to cellular stress including UV radiation. In search for potential mechanisms by which UV light induces ceramide generation, several studies recently reported a pivotal role for hydrolysis of membrane sphingomyelin by ASMase in various cell lines (17 , 19 , 20) . Although activation of ASMase by UV is independent of caspase 8 (19) or a nuclear signal (20) , upstream events leading to ASMase activation remain unknown as well as the specific molecular mechanisms mediating this activation.

In a recent study, we demonstrated that activation of ASMase in response to phorbol esters proceeds through PKC{delta}-dependent phosphorylation at serine 508 (1) . Therefore, the purpose of the current study was to evaluate the role of PKC{delta} in activation of ASMase via phosphorylation in response to UV stimulation and to determine the role of the ASMase pathway in UV-induced apoptosis. Here, the results from this study show that UV radiation activates ASMase in a PKC{delta}-dependent manner. Targeting PKC{delta} by RNA interference or its inhibition by rottlerin blocked UV light-induced ASMase phosphorylation and membrane translocation. Furthermore, UV light failed to induce a ceramide response in MCF-7 cells expressing ASMaseS508A mutant, which functioned as a dominant negative regulator of ASMase activation. In contrast to ASMaseWT cells, ASMaseS508A overexpressors were markedly protected from UV light–induced mitochondrial injury, cytochrome c release, and caspase activation. These findings reveal a novel role for ASMase phosphorylation in cellular stress response to UV radiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
RPMI media and other cell culture material such as FBS were from Invitrogen. Bovine sphingomyelin for SMase assays was from Avanti polar lipids (Alabaster, AL, USA). [Choline-methyl-14C]sphingomyelin was kindly provided by Dr. Alicja Bielawska (Medical University of South Carolina, Charleston, SC, USA). Antibodies against Bax, Bax 6A7, and cytochrome c were from BD Biosciences (San Jose, CA, USA). HSP-60 polyclonal antibody, PKC{delta} polyclonal antibody, and horseradish peroxidase (HRP)-conjugated antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal caspase 9 antibody was from Cell Signaling (Danvers, MA, USA). Monoclonal antibodies against V5 epitope and fluorescently conjugated secondary antibodies were from Invitrogen (Carlsbad, CA, USA). Mitotracker Green FM dye was from Molecular Probes. Antiphosphoserine (polyclonal) antibody was from Zymed (San Francisco, CA, USA). Fatty acid free albumin and all other materials were from Sigma (St. Louis, MO, USA).

Cell lines and culture conditions
MCF-7 cells were originally obtained from ATCC (Manassas, VA, USA). Cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. Testing for presence of mycoplasma infections was done routinely on a monthly basis.

p-ASMase peptide and antisera production
The p-ASMase antiserum was raised at the Sigma Genosys facility. Briefly, two peptides were synthesized, peptide 1:DGNYSRS[pSer508]HVVLDHC and peptide 2: DGNYSRSSHVVLDHC. The integrity of the peptides was verified by HPLC and mass spectroscopy. Peptides were keyhole limpet hemocyanin (KLH) conjugated via added cysteine residue at the C-terminal region. Two rabbits were used for immunizations. Third bleed serum was double affinity purified by consecutive elution through two columns using peptides 1 and 2. ELISA testing was performed on the crude, flow-through, and eluant from both columns. Specificity of the antibody was additionally demonstrated using peptide 1, which competed for the p-ASMase epitope in immunofluorescence and Western blotting experiments.

UV irradiation
MCF-7 cells grown on 10 cm dishes or 2 cm poly-L-lysine coated confocal dishes were covered with a thin layer of PBS before irradiation. Radiation was performed using GS Gene Linker UV chamber (Bio-Rad, Hercules, CA, USA), which emits UVC light ({lambda}avg=254 nm) at a dose of 50 joules/m2. Cells were then reincubated with RPMI media and then collected for analysis at the indicated time(s) postirradiation.

SMase assays
In vitro enzymatic assays for sphingomyelinases were performed using [choline-methyl-14C]sphingomyelin. The assays were performed as previously reported (28 , 29) .

RNA interference
The gene silencing of PKC{delta} was performed essentially as described previously (1) using Oligofectamine transfection vehicle from Invitrogen. Sequence specific siRNA reagents were purchased from Qiagen (Valencia, CA, USA). The target sequence of the designed siRNA is AAC GAC AAG ATC ATC GGC AGA. The specificity of the RNA interference (RNAi) was verified by sequence comparison with the human genome database using the NIH blast program.

Western blotting
MCF-7 cells subjected to the indicated treatments were scraped on ice-cold PBS and collected by centrifugation. Cells were lysed by sonicating in RIPA lysis buffer containing the following mixture of protease and phosphatase inhibitors: 5 mM sodium fluoride, 1.75 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 20 mM β-glycerol phosphate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 µM microcystin. Lysates were normalized to protein concentration using Bio-Rad Bradford reagent (Bio-Rad). Samples (30 µg) were boiled in Laemmli sample buffer and separated by SDS-PAGE. This was followed by protein transfer to 0.45 µm nitrocellulose membranes and blocking with 5% milk or bovine albumin solution. Blots were probed by overnight incubation with the indicated antibodies. Membranes were washed three times with 0.1% Tween 20 in Tris-buffered saline before the addition of the appropriate HRP conjugated secondary antibodies. The ECL immunoblotting detection system (Amersham Biosciences, Piscataway, NJ) was used to visualize the bands. Equal loading was checked by probing with actin specific antibody (Santa Cruz).

Confocal microscopy analysis
Approximately 5 x 105 cells were seeded to a 2 cm poly-L-lysine coated confocal plate (MatTek Corp, Ashland, MA, USA). After treatment, cells were fixed with 4% paraformaldehyde solution for 10 min and permeabilized with methanol for 5 min. Blocking was done in 2.5% FBS solution. Primary antibodies were diluted in a solution of 1.5% FBS/0.15% saponin and incubated for 3 h. The samples were then washed with 1.5% FBS solution three times. This was followed by incubation with secondary antibodies for 1 h at room temperature. Samples were stored at 4°C until image acquisition. Images were acquired using a Zeiss Laser scanning confocal microscope (LSM 510). Excitation wavelengths of 488, 543, and 633 were used. Images were acquired at equatorial planes of monolayer cells guided by DRAQ5 (Alexis) nuclear staining.

Cell fractionation
After treatment and collection, cells were lysed in a buffer consisting of 300 mM sucrose, 20 mM Tris pH 7.4, 2 mM EDTA, 2 mM MgCl2, 1.75 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 µM microcystin. After incubation on ice for 30 min, cells were disrupted by repeated passage through a 27 G needle and centrifuged in an Eppendorf centrifuge at 1000 g (10 min, 4°C) for collection of nuclei and then at 10,000 g (10 min, 4°C) for collection of heavy membranes containing mitochondria and lysosomes. The last step was centrifugation at 100,000 g in an ultracentrifuge (10 min, 4°C) to obtain cytosolic components in the supernatant.

In vivo analysis of mitochondrial network complexity
Mitochondrial network complexity was analyzed using a previously described fluorescence recovery after photobleaching (FRAP)-based assay (30 , 31) . Briefly, mitochondrial networks of MCF-7 cells were labeled with the lipophillic dye Mitotracker Green FM (Molecular Probes, Eugene, OR, USA) at a concentration of 25 nM. After 30 min, the medium was changed, and fresh medium was added. Cells were placed in a closed chamber, under controlled temperature and CO2 settings, fitted on a Zeiss LSM510 laser scanning confocal microscope. Bleaching of mitochondrial fluorescence was done at random regions of interest (ROIs) of equal size (10 µm diameter). Recovery of fluorescence to the photobleached ROIs was measured every 0.4 s over a period of 40 s. Values were normalized to the prebleach fluorescence intensity of each ROI and plotted vs. time.

Mass spectroscopy for ceramide and sphingomyelin
Sphingolipids were extracted from the different treatment samples by the Bligh Dyer technique. Sphingolipid analysis was performed using a previously described electrospray ionization/tandem mass spectrometry (ESI-MS/MS) on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer, operating in a multiple reaction monitoring positive ionization mode. This method has been recently described (32) .

Plasmid constructs and overexpression
The V5 tagged ASMaseWT and ASMaseS508A plasmids were previously described (1) . Both plasmids have full-length ASMase cDNA ligated into pEF6/V5-His-TOPO vector (Invitrogen) with the V5 epitope situated at the C terminus of ASMase. For stable overexpression experiments, endotoxin-free plasmids were transfected into MCF-7 cells using Effectene (Qiagen) according to the manufacturer’s recommendations. After 48 h, selection antibiotic (blasticidin, 10 µg/ml) was added. Cells were allowed to grow for 5 days before splitting 1/10 dilution into 10 cm dishes. After 1 wk cells were split into 96-well plate at a density of 2 cells/well with antibiotic concentration now reduced to 7 µg/ml. Growth of potential stable overexpressors was monitored by light microscopy, and ~10 clones were isolated from each 96-well plate. Stable overexpression was verified by confocal microscopy as well as Western blotting.

Statistical analysis
Mann-Whitney or Student’s t tests were performed between control and treated states and/or between treatment and treatment plus RNAi-mediated inhibition states on a minimum of three independent experiments. A P value of 0.05 or less was considered as statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Modulation of ceramide and sphingomyelin species by UVC in a PKC{delta}-dependent manner
Ceramide generation occurs as an early event during the cellular stress response to UV radiation (19 , 33 , 34) . To evaluate changes in particular ceramide species, MCF-7 cells were irradiated with UVC (50 J/m2) and collected at 5, 10, 15, 30, and 60 min after exposure. Sphingolipids were extracted according to the Bligh Dyer method and analyzed by LC/MS as described previously (32) . As shown in Fig. 1 A, UVC induced significant elevation in C16-ceramide (1.5-fold) and C24:1- ceramide (2-fold) within 5 and 30 min, respectively. There were no significant changes in C24 and C14 ceramides (Fig. 1A ) or other minor ceramide species (C20, C18, and C18:1 ceramides, data not shown) along this time course. Importantly, mass spectrometric measurements of sphingomyelin (SM) species revealed that the observed changes in C16-ceramide levels were paralleled by a transient drop in C16-SM levels (Fig. 1B ). Other SM species such as C24, C24:1, and C14 SM (Fig. 1B ) or C20, C18, and C18:1 SM (data not shown) had no significant changes. These results suggested activation of a sphingomyelinase acutely after UV radiation of MCF-7 cells.


Figure 1
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  		Figure 1. Modulation of ceramide and sphingomyelin species by UV radiation. MCF-7 cells were seeded in 100 mm dishes (5x105 cells/plate). Cells were coated with a thin layer of PBS and exposed to UVC radiation (50 J/m2) with dish lids off. Cells were collected at 5, 10, 15, 30, and 60 min after radiation and sphingolipids were extracted by the Bligh Dyer technique. Ceramide species (A) and sphingomyelin species (B) were analyzed by mass spectroscopy, and each sample was normalized to its respective total phospholipids content. Shown are averages of 3 determinations ± SE.

Although several studies converged on a central role for ASMase during UVC-induced cellular stress, mechanisms regulating ASMase remain unclear. In a recent study, we reported that activation of ASMase proceeds through PKC{delta}-mediated phosphorylation at serine 508 (1) . To further understand the upstream events by which UV light activates ASMase, cell lines stably overexpressing WT-ASMase (ASMaseWT cells) or ASMase S508A mutant (ASMaseS508A cells) were generated. In vitro ASMase activity of both cell types was evaluated at 5, 10, and 60 min postradiation using [choline-methyl-P14PC]sphingomyelin as a substrate. The data showed that in ASMaseWT cells there was a transient activation of ASMase peaking at 10 min (2-fold) after UV light exposure. However, within the same time frame, the enzymatic activity of ASMaseS508A cells remained unchanged (Fig. 2 A). To determine the importance of ASMase activation in regulating the ceramide response to UV light, the levels of ceramide were analyzed in ASMaseWT and ASMaseS508A cells basally and at 5, 10, 30, and 60 min after radiation. As shown in Fig. 2B , ceramide levels were transiently albeit significantly elevated at 5 and 10 min after exposure of ASMaseWT cells to UVC. In contrast, ASMaseS508A cells failed to trigger a ceramide response (including C24:1 ceramide) to UV radiation, suggesting that the C24:1 ceramide response is dependent on production C16-ceramide. This is in line with the previous finding that the ASMaseS508A mutant serves as a dominant negative form of ASMase, overexpression of which is capable of blocking ceramide formation from endogenous ASMase in mammalian cells (1) . Therefore, taken together, these findings indicate that the ceramide response after UVC radiation is secondary to ASMase activation in MCF-7 cells.


Figure 2
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  		Figure 2. Activation of the ASMase/ceramide pathway by UV light. MCF-7 cells stably overexpressing ASMaseWT or ASMaseS508A were plated on 100 mm dishes (5x105 cells/plate). After UV radiation (50 J/m2), cells were collected at indicated time points. A) In vitro SMase assay was performed in an acidic buffer (pH 4.5) using 14C radiolabeled sphingomyelin as a substrate. Results were normalized to amount of proteins used (100 µg/sample). *Statistically significant differences. B) ASMaseWT and ASMaseS508A cells were collected 5 and 30 min post-UV exposure. Ceramide levels were measured by mass spectroscopy as described under Materials and Methods and normalized to total phospholipids. Results shown are averages of 3 experiments ± SE. *Significant differences at same time point between the 2 cell types; {Delta}significant differences in ASMaseWT cells in comparison to basal levels (unpaired t test, P<0.05).

Based on our recent work (1) and the above results, it became important to investigate whether UV light activates ASMase through serine phosphorylation. To that end, the effects of UV on ASMase phosphorylation were examined in MCF-7 cells by Western blotting using a recently developed phospho-specific antibody to Ser-508 of human ASMase (described in Materials and Methods). Transient time-dependent phosphorylation of ASMase at S508 was detected within 5 min of UV radiation (Fig. 3 A). Importantly, phosphorylation of ASMase occurred in concert with the increased in vitro activity and with ceramide generation observed earlier. This result was further corroborated by immunofluorescence studies whereby UVC radiation was observed to stimulate ASMase phosphorylation as evidenced by the increased fluorescence using the p-ASMase antibody. In addition, p-ASMase was observed to relocate at least in part to the plasma membrane albeit with strong punctate cytosolic staining (Fig. 3B ). Notably, pretreatment with the PKC{delta} inhibitor, rottlerin, or transfection of PKC{delta}-specific RNAi blocked the p-ASMase response to UV radiation.


Figure 3
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  		Figure 3. UV light induces phosphorylation of ASMase at S508. A) Lysates (30 µg) of MCF-7 cells collected at the indicated times post-UV radiation were separated by SDS-PAGE. Levels of p-ASMase were determined by Western blotting using p-ASMase (S508) polyclonal antibody described under Materials and Methods. B) Subcellular localization of p-ASMase as detected by indirect immunofluorescence. Cells plated on poly-L-lysine coated 2 cm dishes (50–60% confluence) were pretreated or not with the PKC{delta} inhibitor rottlerin or PKC{delta}-specific RNAi (5 nM, 48 h) before UV exposure. Staining for p-ASMase was detected in the green channel (Alexa Fluor 488), and nuclei were labeled with DRAQ 5 nuclear dye. C) Effect of PKC{delta} knockdown on UV-induced ASMase phosphorylation. Cells were transfected with 5 nM of either SCR or PKC{delta} specific RNAi for 48 h. Immunoprecipitates from each sample were loaded to 10% SDS gels and probed with anti phosphoserine antibody. PKC{delta} was detected at 78 KDa using a specific polyclonal antibody. Results shown represent 3 independent experiments. CN = control.

To investigate whether this phosphorylation is mediated by PKC{delta} as recently reported (1) , MCF-7 cells were transfected with 5 nM of scrambled (SCR) or PKC{delta} specific RNAi sequences. After 48 h, when maximal loss of PKC{delta} protein (>70%) was observed by Western blotting, cells were irradiated with UVC (50 J/m2, 5 min) or treated with phorbol 12-myristate13-acetate (PMA; 100 nM, 1 h) as a positive control. As shown in Fig. 3C , serine phosphorylation of ASMase was evident in the SCR transfected group under both treatment conditions; however, this response was lost after knockdown of PKC{delta}. Taken together, these results indicate that UV light induces ASMase phosphorylation at S508 through a PKC{delta}-dependent mechanism.

Overexpression of ASMaseS508A rescues MCF-7 cells from UV-induced mitochondrial fragmentation and cytochrome c release
Mitochondrial fragmentation is a shared morphological feature during cellular stress responses (35) . Recently, several studies have suggested an important role for ceramide as a regulator of mitochondrial outcomes (36 37 38 39 40) . The differential ceramide response observed in ASMaseWT and ASMaseS508A cells prompted us to investigate mitochondrial responses of the same cells after UV radiation. To that end, mitochondrial shape of control (LacZ), ASMaseWT, and ASMaseS508A MCF-7 cells was examined by staining with the mitochondrial heat shock protein, HSP-60. As shown in Fig. 4 A, under basal conditions, mitochondria of LacZ, ASMaseWT, and ASMaseS508A cells had essentially elongated tubular morphology. After exposure to UV light, 58 ± 3% of LacZ cells and 61 ± 2% of ASMaseWT cells had short and fragmented mitochondria. In contrast, only 31 ± 4% of ASMaseS508A cells showed mitochondrial fragmentation after UV radiation. Changes in the mitochondrial morphology induced by UV light were further evaluated by fluorescence recovery after photobleaching (FRAP), a technique previously described by Youle and coworkers (30 , 31) . After photobleaching, fluorescence recovery of a lipophilic mitochondrial dye (Mitotracker Green FM) was observed over a 40 s time frame in MCF-7 cells before and after UV radiation. Overall, UV radiation caused marked reduction in fluorescence recovery of all cell types (Fig. 4B ), indicative of mitochondrial fragmentation. Notably, both LacZ and ASMaseWT cells showed ~40% reduction in their ability to recover fluorescence after bleaching whereas ASMaseS508A cells had only 20% reduction, further confirming the observed differences in mitochondrial morphologies (Fig. 4B ).


Figure 4
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  		Figure 4. Role of ASMase in UV-induced mitochondrial fragmentation. A) MCF-7 cells stably transfected with ASMaseWT, ASMaseS508A, or control plasmid (LacZ) were plated on poly-L-lysine coated 2 cm confocal dishes. Cells were irradiated with UVC and incubated for 6 h before immunofluorescence experiments. Mitochondrial morphology was examined using the mitochondrial marker HSP-60 (Alexa Fluor 488: green channel). Nuclei were labeled with DRAQ5. At least 100 cells from radiated and control groups were examined in 3 independent experiments. *Significant differences in comparison to UV-irradiated Lac Z cells (unpaired t test, P<0.05). B) Effect of UV light on mitochondrial network continuity. Cells treated as in A, were labeled with Mitotracker Green dye for 30 min. Analysis of mitochondrial network complexity was done using a FRAP-based assay described under Materials and Methods. Results were normalized to fluorescence intensity observed at t = 0 from regions of interest. Similar values were obtained in 3 independent experiments.

Since mitochondrial shape changes were previously linked to the mitochondrial pathway of apoptosis, the distribution of cytochrome c was examined by immunofluorescnece. Redistribution of cytochrome c from the mitochondria to the cytosol and nucleus was observed 6 h after UV radiation, indicating commitment to apoptosis (Fig. 5 A). Quantitatively, cytochrome c release was seen in 40 ± 4% of LacZ cells, 48 ± 3% of ASMaseWT cells, but only in 19 ± 3% of ASMaseS508A cells (Fig. 5A ). Additionally, a differential centrifugation approach revealed that ASMaseS508A cells had a substantially less amount of cytochrome c in their cytosolic fractions after UV stress (Fig. 5B ). Taken together, these results implicate ASMase in transducing, at least in part, the effects of UVC on mitochondrial morphology and dynamics.


Figure 5
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  		Figure 5. ASMaseS508A are protected from UV-induced cytochrome c release. MCF-7 cells adherent on poly-L-lysine coated dishes were irradiated with 50 J/m2 of UVC light. A) Fixation and immunofluorescence processing was carried 6 h later. Cytochrome c was stained using a specific monoclonal antibody (red channel). Nuclei were counterstained with DRAQ5 dye. B) Cells of the indicated treatment groups were fractionated, and cytosolic extracts were isolated as discussed in Materials and Methods. Fractions were analyzed for levels of cytosolic cytochrome c by Western blotting. Data represent means from 3 independent experiments ± SD each with 100 cells per cell type. *Significant differences in comparison to UV-irradiated Lac Z cells (unpaired t test, P<0.05).

Resistance of ASMaseS508A cells to UV-induced bax activation
The above results then suggested a role for ASMase upstream of mitochondrial injury in response to UVC. A critical event during the cellular stress response to UV radiation is the conformational change and mitochondrial translocation of Bax (41) . Insertion of Bax into mitochondrial membranes has been associated with mitochondrial outer membrane permeabilization (MOMP), mitochondrial fragmentation, as well as opening of the permeability transition pore (PTP; refs. 42 43 44 ). To evaluate Bax activation, healthy and UV-irradiated MCF-7 cells were costained with the mitochondrial marker HSP-60 and with Bax 6A7 monoclonal antibody. The Bax 6A7 antibody specifically recognizes active Bax after its conformational change and insertion into the mitochondrial outer membrane (45 , 46) . As shown in Fig. 6 , activation of Bax and its insertion into mitochondrial membranes could be detected in 49 ± 3% of LacZ cells and 53 ± 3% of ASMaseWT cells. Importantly, only 24 ± 3% of ASMaseS508A cells contained active Bax in their mitochondria. Together, these findings indicate that the inability of ASMaseS508A cells to generate ceramide in response to UV radiation confers protection from mitochondrial damage.


Figure 6
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  		Figure 6. Differential Bax activation in ASMaseWT and ASMaseS508A cells. MCF-7 cells (LacZ, ASMaseWT, and ASMaseS508A) were seeded on poly-L-lysine coated confocal dishes at 50–60% confluency. Samples were processed for immunofluorescence 6 h after exposure to UV light. Active Bax was visualized using conformation specific Bax 6A7 monoclonal antibody and mitochondria were marked using HSP-60 staining. Data are mean ± SD from 3 independent experiments each with 100 cells per cell type. *Significant differences in comparison to UV-irradiated Lac Z cells (unpaired t test, P<0.05).

Resistance of ASMaseS508A cells to UV-induced apoptotic cell death
To determine whether activation of ASMase by phosphorylation at S508 contributes to UV-induced cell death, we analyzed caspase 9 and poly (ADP-ribose) polymerase (PARP) cleavage, two hallmarks of the apoptotic cascade. Western blotting experiments conducted in LacZ and ASMaseWT cells showed substantial PARP cleavage and the presence of the active cleaved form of caspase 9 at 24 h after radiation. In contrast, moderate PARP and caspase 9 activation were detected in ASMaseS508A cells under the same conditions (Fig. 7 A). It is worth noting here that MCF-7 cells are caspase 3 null (47) , and therefore activation of this executioner caspase was not pursued. To further confirm a role for ASMase S508 phosphorylation in the response to UV light, a flow cytometry approach was utilized to quantitate dead cells showing positive annexinV/PI staining (top right quadrant). As shown in Fig. 7B , within 12 h, UV radiation resulted in 20 and 27.5% dead cells in LacZ and ASMaseWT cells, respectively, whereas the same treatment of ASMaseS508A cells resulted in 3.4% cell death. After 24 h, 42% of LacZ cells and 46% of ASMaseWT cells but only 25% of ASMaseS508 cells were killed by UV stress (Fig. 7B ). These findings provide evidence for phosphorylation of ASMase at S508 as part of the apoptotic cascade elicited by UV radiation.


Figure 7
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  		Figure 7. Resistance of ASMaseS508A cells to UV-induced apoptosis. A) Caspase-9 activation and poly (ADP-ribose) polymerase cleavage were studied by Western blotting 24 h post-UV exposure. Protein loading was checked by probing for β-actin. B) After 12 and 24 h, apoptosis of the three cell types was quantified by annexin V/propidium iodide staining followed by flow cytometry. Annexin V and PI values for each treatment were plotted on the x axis and y axis respectively. Shown are % cells of each treatment group in top right quadrant (annexin V+, PI+). Data represent 3 independent experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Elimination of UV-damaged cells by apoptosis constitutes an important antitumorigenic innate response. This study investigated activation of ASMase by a PKC{delta}-dependent mechanism involving phosphorylation of ASMase at a specific serine residue during UV light-induced apoptosis. The data presented here show that activation of ASMase by UV light is paralleled by an increased phosphorylation at serine 508. Targeting PKC{delta} by RNA interference and pharmacologic inhibitors blocked ASMase phosphorylation and plasma membrane translocation induced by UV light. Additionally, cells overexpressing a nonphosphorylatable form of ASMase (ASMaseS508A) had no significant change in either ASMase activity or ceramide levels after UV exposure, demonstrating that this mutant functions as a dominant negative and that the phosphorylation of Ser-508 is necessary for UV-induced ceramide. Although others have implicated ASMase in UV-induced apoptosis, this is the first report to identify PKC{delta} as a key upstream kinase. In contrast to ASMaseWT cells, ASMaseS508A cells were markedly protected from UV-induced mitochondrial fission. In ASMaseWT cells, Bax activation and cytosolic cytochrome c release were more than double the response seen in ASMaseS508A cells after UV radiation. These findings are of major importance as ASMaseS508A cells were resistant to UV-induced apoptosis.

An important conclusion of the current study emanates from defining a role for PKC{delta} upstream of ASMase activation during UV light-induced cellular stress response. Several recent studies converged on a central role for ASMase in mediating the ceramide response following UV radiation in lymphocytic and cervical cancer cell lines (17 , 19 , 20 , 48) . However, little is known about upstream mechanisms leading to ASMase activation by UV light or other stimuli. The results presented herein indicate that activation of ASMase by UV light proceeds through PKC{delta}-dependent phosphorylation. First, UV radiation induced a transient time dependent phosphorylation of ASMase at S508 paralleled by elevation of in vitro enzymatic activity. Second, knock down of PKC{delta} blocked serine phosphorylation of ASMase after UV exposure. Third, pharmacologic inhibition of PKC{delta} with rottlerin blocked translocation of ASMase to the plasma membrane. Fourth, in contrast to ASMaseWT cells, ASMaseS508A cells retained basal enzymatic activity and ceramide levels after UV radiation. Although several studies have converged on a pivotal role for PKC{delta} during UV-induced apoptosis, the molecular mechanisms underlying this process are still under current investigation. The current results therefore bring together these two important pathways that mediate/regulate stress and apoptotic responses through identification of ASMase as a direct effector of PKC{delta} during the UV response.

The findings of the current study also highlight an important function for the PKC{delta}/ASMase/ceramide pathway in regulating mitochondrial responses to UV radiation. Morphologically, mitochondria of cells overexpressing dominant negative ASMaseS508A (1) were markedly protected from UV light-induced mitochondrial fragmentation seen in LacZ and ASMaseWT cells. This finding is of particular importance, as breakdown of the tubular mitochondrial network to more fragmented organelles is currently regarded as an early event during the apoptotic cascade seen in mammals and yeast (35 , 49 , 50) . Molecular mechanisms underlying mitochondrial fragmentation during apoptosis are not fully understood. However, considerable evidence points to the involvement of Bax, a proapoptotic member of the Bcl-2 family, and members of the mitochondrial fission machinery such as DRP1 (dynamin related protein) and Fis1 in mitochondrial splitting (31 , 42 , 51 52 53) . Consistent with this model, the current results indicate that blocking of the ASMase/ceramide pathway rescues MCF-7 cells from UV-induced Bax oligomerization on OMM. This finding affords a potential explanation for the differential mitochondrial morphology of ASMaseWT vs. ASMaseS508A cells discussed earlier. Additionally, ASMaseS508A cells were protected from cytosolic cytochrome c release and consequent caspase 9 activation and PARP cleavage post-UV radiation. It is worth noting here that mitochondrial fission has been reported to be an obligate step upstream of cytochrome c release during some cellular death stimuli (54) . Reciprocally, inhibition of mitochondrial fission such as by down-regulation of Fis1 or overexpression of dominant negative Drp1 markedly abrogates cytochrome c release and cell death (51 , 53 , 54) .

Several studies have recently addressed cellular mechanisms by which ceramide generation induces mitochondrial dysfunction (36 , 38 , 55) . As a result of the cumulative work of those studies, three models are currently proposed. First, ceramide could induce activation of proapoptotic members of the Bcl-2 family such as Bid and Bax leading to MOMP (17 , 56 57 58) . Second, ceramide could induce formation of channels larger than 10 nm in diameter in mitochondrial membranes allowing the release of proteins up to 60 KDa (59) . Third, ceramide could induce opening of the PTP (60) . The work presented here draws a fourth connection in which ceramide generation during cellular stress response triggers mitochondrial fission. Although this could be explained in part by the effect of ceramide on Bax conformational change, one could not exclude at this stage a potential effect of ceramide on other mitochondrial fission effectors such as DRP1 and Fis1. Another intriguing link emanates from previous studies characterizing ceramide as an inhibitor of the phospholipase D (PLD) reaction (61 62 63 64) . Importantly, recent studies uncovered involvement of a specific mitochondrial PLD activity (Mito PLD) in fusing mitochondrial membranes (65) . Therefore, it is conceivable that inhibition of Mito PLD by ceramide affords an alternative explanation for the current findings. Elucidation of such possibilities needs to be addressed in future work.


   ACKNOWLEDGMENTS
 
We thank the Lipidomics Core Facility (J. Bielawski and A. Bielawska) at the Medical University of South Carolina. Y.H.Z. thanks K. Norris (NIH) for advice with mitochondrial network analysis assay and Yi Te Hsu for carefully reading the manuscript. This work was supported by the National Cancer Institute grant NCI P01-CA97132 (to Y.A.H.) and a MERIT Award to L.M.O. by the Office of Research and Development, Department of Veterans Affairs, Ralph H. Johnson VA Medical Center, Charleston, South Carolina. The work at the Lipidomics Core is supported by NIH C06 RR018823. Y.H.Z. is supported in part by the CFRI New Horizons Fund.

Received for publication May 7, 2007. Accepted for publication July 12, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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