REVIEW Ceramide Regulates Cellular Homeostasis Via Diverse Stress

Home , Acid sphingomyelinase, Ceramide, Ceramide kinase

Ceramide regulates cellular homeostasis via diverse stress signaling pathways PP Ruvolo

University of Florida Shands Cancer Center and The Department of Medicine, Gainesville, Florida, 32610–0232, USA

The sphingolipid ceramide is an important second signal mol- system exists whereby membrane building blocks and growth ecule that regulates diverse signaling pathways involving agonists are produced during conditions favorable to cell apoptosis, cell senescence, the cell cycle, and differentiation. For the most part, ceramide’s effects are antagonistic to growth growth. In contrast, stress stimuli promote the breakdown of and survival. Interestingly, ceramide and the pro-growth agon- sphingomyelin by phospholipases known as sphingo- ist, diacylglycerol (DAG) appear to be regulated simultaneously myelinases (SMase) and ceramide is produced. Like DAG, cer- but in opposite directions in the sphingomyelin cycle. While amide has emerged as an important second messenger mol- ceramide stimulates signal transduction pathways that are ecule, except that ceramide-mediated signaling usually associated with cell death or at least are inhibitory to cell 5–11 growth (eg stress-activated protein kinase, SAPK, pathways), involves stress pathways. Since at least one enzyme DAG activates the classical and novel isoforms of the protein (sphingomyelin synthase) appears to regulate DAG and cera- kinase C (PKC) family. These PKC isoforms are associated with mide simultaneously but in opposite directions, it is possible cell growth and cell survival. Furthermore, DAG activation of that DAG and ceramide have counterbalancing effects on cell PKC stimulates other signal transduction pathways that sup- growth and apoptosis.12 Consistent with the notion that DAG port cell proliferation (eg mitogen-activated protein kinase, MAPK, pathways). Thus, ceramide and DAG generation may and ceramide have counterbalancing effects, DAG can attenu- 13 serve to monitor cellular homeostasis by inducing pro-death or ate the effects of ceramide and ceramide can antagonize pro-growth pathways, respectively. The production of ceramide DAG by inhibiting PKC.14–16 An elegant homeostatic regulat- is emerging as a fixture of programmed cell death. Ceramide ory system is emerging with DAG as a growth positive regu- levels are elevated in response to diverse stress challenges lator and ceramide as a negative growth regulator. Since each including chemotherapeutic drug treatment, irradiation, or treatment with pro-death ligands such as tumor necrosis factor effector is able to transmodulate the signaling pathways regu- ␣,TNF␣. Consistent with this notion, ceramide itself is a potent lated by the other, it is possible that net expression ofDAG apoptogenic agent. Ceramide activates stress-activated protein vs ceramide may create a sort ofcellular rheostat. 13 It is poss- kinases like c-jun N-terminal kinase (JNK) and thus affects tran- ible that the dominant second signal molecule produced dur- scription pathways involving c-jun. Ceramide activates protein ing growth (ie DAG) or stress (ie ceramide) conditions could phosphatases such as protein phosphatase 1 (PP1) and protein phosphatase 2 (PP2A). Ceramide activation of protein phospha- promote signal pathways that determine post-translational tases has been shown to promote inactivation of a number of modification ofpotential ‘survival sensor’ molecules (eg pro-growth cellular regulators including the kinases PKC ␣ and Bcl217). Akt, Bcl2 and the retinoblastoma protein. A new role has Ceramide is produced during diverse stress stimuli includ- recently emerged for ceramide in the regulation of protein syn- ing chemotherapeutic drug treatment,18–20 TNF ␣ treatment,21 thesis. Ceramide-induced activation of double-stranded RNA- 22 23 dependent protein kinase (PKR), a protein kinase important in ischemia/reperfusion, FAS antigen activation, cortico- 24 24 anti-viral host defense mechanisms and recently implicated in steroid treatment, or irradiation. The generation ofceram- cellular stress pathways, results in the inhibition of protein syn- ide is so common during apoptosis that it has been considered thesis as a prelude to cell death. Taken together, these proper- a universal feature of this process.1,3 It is becoming increas- ties of ceramide suggest that this important second-signal mol- ingly clear that ceramide is a key regulator ofstress signaling ecule may have useful properties as an anti-neoplastic agent. Thus, strategies to promote ceramide metabolism or use of cer- pathways involved in apoptosis signaling, cell cycle control, 5–11 amide analogs directly may one day become useful in the treat- cellular senescence, and cell differentiation. Ceramide ment of diseases like leukemia. Leukemia (2001) 15, 1153–1160. activates a number ofkinases (Figure 1) including stress-acti- Keywords: ceramide; apoptosis; PP2A; JNK; RAX; stress signal vated protein kinases (SAPKs) such as the jun kinases pathway (JNKs25–27), kinase suppressor ofRas (KSR 28–30), and the atypi- cal PKC isoform, PKC ␨.31–33 Conversely, ceramide can inhibit kinases such as the classical and novel PKC isoforms (eg PKC Introduction ␣14–16) and the PKB kinase, Akt.29,34–36 The mechanism ofkinase inhibition appears to involve ceramide’s ability to activate Ceramide is a naturally occurring sphingolipid that is a key 1 protein phosphatases (Figure 1 and Refs 16, 17, 36–39). The component in the sphingomyelin cycle. In this capacity, cer- ceramide-activated protein phosphatase (CAPP) is probably a amide serves an important role in membrane structure. During member(s) ofthe PP2A family 37,38 although ceramide can acti- growth conditions, ceramide and phosphatidylcholine are vate PP1 as well.39 Ceramide activation ofprotein phospha- converted to sphingomyelin by sphingomyelin synthase.1 A tases not only suppresses survival signaling pathways by inac- byproduct ofthis reaction is diacylglycerol (DAG), a potent tivation ofkinases, but also directly dephosphorylate protein kinase C (PKC) activator.2 Since DAG-mediated PKC substrates directly involved in cell survival (eg Bcl217). This signaling pathways tend to support cell survival,3,4 an elegant review will focus on ceramide as a stress second signal molecule and the stress signaling pathway this important sphingolipid regulates including a novel pathway that regulates Correspondence: P Ruvolo; Fax: 352-846-1193 protein synthesis. Received 20 March 2001; accepted 6 April 2001 Review PP Ruvolo 1154 mouse models has enabled investigators to monitor the effect ofSMase functionon apoptosis. 45,48 The sphingomyelin pathway for ceramide generation demonstrates the versatility of effector responses that can be generated by ceramide. In fact, divergent responses from a single receptor may be generated depending on whether neutral SMase or acidic SMase is involved.41 Signaling pathways mediated by the TNF 55 kDa receptor (p55) may result in an inflammatory response or the induction ofapoptosis. 41,50–54 To trigger the pro-inflammatory cascade, neutral SMase is coupled to p55 via the FAN protein.52,54 Activation of apoptosis via the TNF receptor involves activation ofacidic SMase through the p55 death domain.53 There is also evidence that neutral SMases participate in apoptotic mechanisms.55–57 For instance, neutral SMase been shown to be involved in hypoxia induced apoptosis in PC12 cells.57 These findings suggest that the level ofregulation ofceramide- mediated responses is complex.

Other molecules involved in ceramide biosynthesis regulate ceramide-mediated pathways

Figure 1 Ceramide activates both protein kinases and protein The influences of other effectors that oppose or support cera- phosphatases. Ceramide activation ofprotein kinases (eg JNK, PKC ␨) mide-regulated signal pathways also seem to be important in and protein phosphatases (eg PP1 and PP2A) allows ceramide to regu- the regulation of the SMase pathway. The effect of DAG, a late multiple stress signaling pathways. Since a number of different molecule that antagonizes ceramide’s actions and is substrates are involved, ceramide is a potent effector molecule. oppositely regulated from ceramide in the sphingomyelin cycle, is discussed above. Another molecule that can appar- Ceramide is generated by multiple metabolic pathways ently antagonize ceramide-mediated signaling is glutathione. It has recently been discovered that neutral SMase is inhibited Considerable interest has developed in ceramide metabolism by glutathione.58 Since glutathione depletion is associated over the past few years. Ceramide is actually a family of highly with apoptotic stresses such as treatment with peroxide or hydrophobic molecules that contain a variable length fatty alkylating agents,59 depletion ofglutathione may be necessary acid (containing 2 to 28 carbons) linked to sphingosine or a for ceramide production.58 This potential regulatory loop links related long chain base.40 Ceramide serves multiple physio- oxidative stress pathways with ceramide-mediated signaling.59 logic roles. Ceramide is an important intermediate in sphingo- Like DAG, effectors that antagonize ceramide action are myelin biosynthesis.1 In addition to its role in the metabolism sometimes byproducts ofceramide metabolism. Deacylation ofmembrane building blocks (ie sphingomyelin), ceramide’s ofceramide by ceramidases yields sphingosine. A number of unique physical properties contribute to membrane structure ceramidases have been identified in various subcellular by influencing membrane lipid ordering and by affecting locations including lysosomes and membranes.60–62 Like cera- membrane porosity.41 In fact, ceramide formation with con- mide, sphingosine exerts divergent effects depending on cell comitant sphingomyelin hydrolysis appears to be necessary type.9 Sphingosine promotes apoptosis in U937 cells27,63,64 for the changes in membrane morphology (eg blebbing, vas- and prostate cancer cells,65 induces cell arrest in Chinese culization, and externalization ofphosphatidylserine) that are hamster ovary cells,66 and promotes proliferation of Swiss 3T3 the hallmark features of the execution phase of apoptosis.42 cells.67 Sphingosine has different effects on stress signaling In addition to its metabolic and structural roles, it is becoming pathways when compared to ceramide. Ceramide promotes increasingly clear that ceramide plays a critical role in trans- Jun phosphorylation while sphingosine does not.27 While membrane signaling, particularly in response to stress chal- sphingosine weakly stimulates SAPK pathways and strongly lenges.5–11 Therefore, it is not surprising that there are multiple inhibits MAPK pathways, ceramide demonstrates the opposite physiologic pathways that produce ceramide. effect.27 Sphingosine is rapidly converted to sphingosine-1 Ceramide can be generated (1) as a byproduct ofsphingo- phosphate (SPP) upon stimulation ofsphingosine kinase by myelin hydrolysis, (2) via de novo pathways involving ceram- growth receptors like nerve growth factor.68 SPP promotes ide synthase, or (3) by the breakdown ofcomplex glycosphin- extracellular signal receptor kinase (ERK) pathways and mito- golipids. Cellular generation ofceramide appears to involve genesis.68–70 Thus, sphingosine can be converted from a some level oftopological control since the various metabolic growth antagonist to an effector that promotes growth. Like- enzymes involved appear to have preferred sub-cellular com- wise, modifications of ceramide also alter effector activity. partments.40 The de novo synthesis ofceramide occurs in the Phosphorylation ofceramide results in a molecule that sup- endoplasmic reticulum (ER) and in mitochondrial mem- ports growth.71 Ceramide kinase produces ceramide1-phos- branes.19,40 Ceramide production during sphingomyelin phate, a molecule which supports cell growth and promotes hydrolysis occurs in multiple locations depending on the DNA synthesis in a manner that is antagonized by ceram- particular SMase involved.43–48 The sphingomyelin pathways ide.72,73 Another mechanism to modulate ceramide’s function have garnered particular attention considering that Niemann- biochemically involves glycosylation. Glycosylation ofceram- Pick disease is the result ofa deficiency in acidic sphingo- ide inactivates the molecule’s negative growth effects and thus myelinase.49 In addition, the availability ofSMase knockout glycosylation may serve as a mechanism for the cell to tolerate

Leukemia Review PP Ruvolo 1155 excess ceramide levels.74 Supporting this notion, an associ- ing PKC ␨.31–33,91 PKC ␨ has been shown to suppress cell ation has been found between multidrug resistance and glyco- growth in human embryonic kidney cells by the activation sylceramide levels in breast cancer cell lines.75 ofSAPK pathways. 33 Expression ofconstitutive active PKC ␨ induced cell cycle arrest while a dominant negative PKC ␨ protein blocked SAPK activation by ceramide.33 PKC ␨ may Ceramide activates protein kinases that are associated with also have a role in inflammatory pathways. While Rac-1 has stress signaling pathways been implicated in JNK activation by ceramide,80 PKC ␨ stimulation by lipopolysacharide activates JNK by a Rac-inde- Ceramide-induced apoptosis often involves the SAPK/JNK sig- pendent mechanism involving PI 3 kinase.91 In addition, PKC naling pathway.76 In U937 cells, the ability ofthe TAM-67- ␨ has been shown to negatively regulate PKB/Akt.92 Since dominant negative c-jun mutant to block ceramide-induced PKB/Akt has been identified as a physiologic Bad kinase,93,94 apoptosis suggested that ceramide initiated a SAPK cascade it is possible that PKC ␨ may regulate ceramide-induced death that resulted in c-jun phosphorylation.76 There are three genes pathways involving Bad. PP2A has also been implicated in that encode JNK (␣, ␤, and ␹) with 12 possible isoforms Akt inactivation35,36 Thus the regulation ofBad pro-apoptotic derived from alternative splicing products.77–79 JNK is acti- function appears to involve both protein phosphatases and vated by diverse stress stimuli,77 while mitogenic stimuli are protein kinases. poor JNK agonists.78 In fact, it appears that ceramide redirects cellular signaling pathways by promoting SAPK cascades at the expense ofMAPK pathways. 27 In addition to the induction Ceramide activates the protein phosphatases PP2A and PP1 ofapoptosis, ceramide activation ofSAPK pathways induces cell cycle arrest and inhibits cell proliferation.33 Thus con- Ceramide is a potent activator ofboth PP2A 37,38 and PP1.39 ditions that favor ceramide generation promote JNK acti- The mechanism ofceramide activation ofthese protein phos- vation. The mechanism by which ceramide activates JNK is phatases is not yet clear. It is known, however, that protein not yet clear. Ceramide has been suggested to activate JNK phosphatases play a critical role in ceramide-mediated pro- via Rac-1,80 PKC ␨,31,33,79,81 and TAK-1.82 It is also unclear cesses. PKC14,16 and Akt35,36 are inactivated by PP2A in which JNK substrates may participate in ceramide-induced response to ceramide. In addition, ceramide-mediated acti- cell death pathways. A number oftranscription factorshave vation ofPP2A also directly targets PKC and Akt substrates. been suggested as downstream targets for ceramide-activated For instance, ceramide promotes PP2A dephosphorylation of JNK including AP-1 and GADD153.8,80,83 However, the Bcl2 resulting in the loss ofBcl2’s anti-apoptotic function. 17 requirement for the participation of these factors in effector- Furthermore, PP2A has recently been shown to directly mediated processes may vary by cell type. For instance, AP- dephosphorylate Bad.95 At least for some members of the Bcl2 1 appears to be required in ceramide-induced apoptosis in family, ceramide activation of PP2A promotes anti-survival human leukemia HL-60 cells and U937 cells.27,83,84 However, signaling by targeting Bcl2 family members directly while c-jun/AP-1 is not essential to sphingosine-induced apoptosis inactivating their physiologic protein kinases.17 in human leukemia U937 cells.27,84 An understanding ofthe Ceramide activation ofPP1 has not been as well studied as potential effects ceramide-activated JNK may have on cell sur- PP2A. Ceramide has been shown to promote dephosphoryl- vival via a transcriptional mechanism(s) will require the identi- ation ofRb in association with growth arrest. 96 Recently, phos- fication of the genes whose expression are affected by ceram- phatidic acid (PA) has been shown to inhibit PP1 but not ide-activated JNK. One candidate that has emerged is PP2A.97 PA was demonstrated to block ceramide-induced cyclooxygenase-2 (Cox285,86). However, while over- dephosphorylation ofRb and PARP cleavage, thus implicating expression ofCox2 has been shown to impede cell cycle pro- PP1 in both cell cycle arrest and apoptotic processes, gression,87 Cox2 inhibits apoptosis.88 Thus downstream JNK respectively.97 targets other than Cox2 are probably involved in apoptotic mechanisms involving ceramide. A potential JNK substrate involved in ceramide-mediated apoptosis is the cellular PKR The complexity ofceramide production and topology allow regulator, RAX.89 Interestingly, ceramide activation ofRAX ceramide to participate in diverse signaling pathways involves protein translation rather than transcription. This novel ceramide regulatory mechanism will be discussed in Since ceramide can be produced by multiple mechanisms in greater detail later. different subcellular compartments, ceramide has the potential to regulate diverse signal transduction pathways. In addition, since there is a fair level of interaction between the Ceramide activates a number ofnon-SAPK kinases various ceramide metabolic pathways and pathways regulated by effectors that transmodulate ceramide action, downstream Ceramide is known to activate a number ofnon-SAPK kinases effects in response to stress will be complex and varied. In including KSR28–30 and PKC ␨.31–33 As described earlier, cera- effect, the dominance of a particular pathway (eg ceramide- mide can mediate either pro-inflammatory or pro-apoptotic mediated anti-growth signaling vs DAG-mediated pro-growth processes.41,50–54 KSR appears to mediate pro-inflammatory signaling) in a cell will probably determine the outcome of responses except when the pro-apoptotic Bcl2 family mem- how that cell responds to the stress challenge. As mentioned ber, BAD, is present.9,29 KSR apparently is required in both above, DAG opposes the effects of ceramide13 and ceramide TNF ␣ and ceramide-induced activation ofERK1/ERK2 in can inhibit PKC.14–16 Ceramide activation ofprotein phospha- intestinal epithelial cells as evidenced in mouse colon cells tase results in PKC ␣ dephosphorylation and inactivation.14,16 expressing kinase-inactive, dominant negative KSR.90 The Inactivation ofPKC ␣ by TNF-mediated signaling can be mechanism ofKSR activation ofERK1/ERK2 involves the blocked by fumonisin B or glutathione and thus suggests a activation ofRaf-1. 90 role for both the de novo pathway and neutral SMase pathway Ceramide appears to mediate multiple mechanisms involv- in TNF-mediated ceramide production.16 The complexity of

Leukemia Review PP Ruvolo 1156 regulation increases as one observes the effects of ceramide DAG) would inﬂuence the function of these proteins at this on the downstream substrates ofPKC ␣. PKC ␣ is a physio- subcellular location. logic Bcl2 kinase.98 Mono-site phosphorylation ofBcl2 at ser- It is more than likely that other mechanisms involving regu- ine 70 is required for Bcl2’s full and potent anti-apoptotic lators of mitochondrial integrity will be affected by alterations function.99,100 Bcl2 is dephosphorylated by its physiologic in ceramide metabolism. The regulation ofapoptosis by signal protein phosphatase, PP2A.101 Ceramide-induced dephos- transduction pathways is complex.104 Many ofthe protein kin- phorylation ofBcl2 via PP2A results in inactivation ofBcl2’s ases and protein phosphatases that participate in these various anti-apoptotic function.17 processes are probably inﬂuenced by ceramide, either directly Recent studies focusing on de novo synthesis ofceramide or indirectly.100 In addition, other signal pathways regulating in the mitochondria have found that carnitine palmitoyl- Bcl2 and other Bcl2 family members are probably regulated transferase I (CPT I) may have a role in ceramide-mediated by ceramide. For instance, the MAPK extracellular-signal apoptosis.102 CPT I facilitates passage of long chain fatty acids regulated kinases (ERKs) ERK1 and ERK2 phosphorylate Bcl2 to the mitochondria. CPT I expression and ceramide pro- at serine 70 under survival conditions.105 As noted above, cer- duction was elevated in murine LyD9 cells deprived ofIL-3 amide inhibits MAPKs including ERK1 and ERK2.27 Important in a fumonisin B-dependent manner.102 Interestingly, CPT I upstream regulators ofthe ERKs including Rafand MEK-1 have apparently can bind Bcl2.103 Perhaps Bcl2 can prevent ceram- been shown to synergize with Bcl2 in blocking cell killing ide synthesis via de novo pathways by sequestering CPT I. induced by factor withdrawal in both human and murine The complexity ofregulation at the level ofjust one cellular hematopoietic cell lines.106,107 It will be interesting to deter- regulatory molecule becomes clear in the example ofcerami- mine if ceramide affects this synergy. Meanwhile, other de’s effects on Bcl2 (Figure 2). In this example, how a cell important regulatory proteins localized in the membranes of expressing Bcl2 weathers a stress challenge would most prob- other organelles and in the plasma membrane would undergo ␣ ably depend on the mitochondrial levels ofPKC , PP2A, CPT similar regulation. Taken as a whole, the capacity ofa cell to I, and other enzymes involved in the de novo ceramide path- survive stress stimuli will probably come down to how well way, etc. In addition, the levels ofceramide produced in the the cell’s survival signaling pathways can weather a challenge mitochondria vs competing levels of antagonistic effectors (eg by ceramide.

Ceramide has a novel role in the inhibition ofprotein synthesis as a prelude to cell death

It is logical that inhibition ofprotein synthesis may precede apoptosis in cells that are challenged by stress responses. Recently, the inhibition ofprotein synthesis has been shown to be a prelude to cell death following growth factor withdrawal in IL-3-dependent murine myeloid cells by a mechanism involving the double-stranded RNA-activated protein kinase, PKR.108 In addition, other stress challenges including treatment with such apoptotic agents as TNF ␣, sodium arsen- ate, and peroxide were shown to promote PKR activation and inhibit protein synthesis.109–112 Since ceramide is suggested as a universal component ofapoptosis 1,6,10 and PKR is activated during diverse stress stimuli, the question arose whether ceramide may regulate PKR. Indeed, a ceramide-mediated SAPK/JNK pathway regulating apoptosis involving a novel mechanism featuring the inhibition of protein synthesis via the activation ofPKR has recently been demonstrated. 89 Activated PKR inhibits protein synthesis by phosphorylating eIF2␣, a molecule required for protein translation initiation events.113–115 Until recently, PKR was known only as a component ofthe host anti-viral defensemechanism where double-stranded viral RNA activated the protein.113–115 To account for non-viral activation of PKR in response to stress, a cellular PKR regulator, RAX, was identiﬁed.112 RAX is acti- Figure 2 Model ofhow ceramide may regulate apoptosis in the vated after stress-induced phosphorylation by an as yet mitochondria. (a) During survival conditions, ceramide production is unidentiﬁed SAPK.112 Since ceramide is a potent activator of suppressed in the mitochondria. PKC ␣ and Bcl2 are functional. Bcl2 SAPKs, it is not surprising that ceramide was found to promote heterodimerizes with and inactivates Bax. The interaction between RAX phosphorylation.89 In addition, ceramide induces protein CPT I and phosphorylated Bcl2 is speculated. (b) During stress con- synthesis inhibition and is more effective at killing cells that ditions, ceramide is produced in the mitochondria by ceramide synthase. PP2A is activated and inhibits PKC ␣ and Bcl2 function. Bax are transfected with RAX as opposed to parental or vector only 89 is free and promotes cytochrome C (cyto C) release from the pore transfected cells. Thus, JNK or another SAPK could possibly complex prior to cell death. mediate some apoptotic mechanisms by acting through RAX.

Leukemia Review PP Ruvolo 1157 Manipulation ofceramide production forchemotherapy in has been shown to augment cell killing ofa number ofcell diseases such as leukemia lines derived from different solid tumors (eg neuroblastoma, lung adenocarcinoma, breast carcinoma) in combination with It is clear that ceramide regulates diverse stress signaling path- the drug, 4-HPR (N-(4-hydroxyphenyl)retinamide).121 In ways that determine the cell’s fate in response to stress stimuli addition, disruption ofceramide metabolism by the sphingo- (Figure 3). Therefore, defects in ceramide metabolism could sine analog, saﬁngol, could similarly potentiate cell killing by potentially affect the cellular response to chemotherapy or 4-HPR in these cell lines and in myelogenous and lympho- other anti-cancer regimens by rendering the cells more resist- cytic leukemia cell lines.121 These promising ﬁndings suggest ant to cell killing, and as has been suggested, could contribute that targeting ceramide metabolism may be useful in future to multi-drug resistance.116 TNF ␣-resistant117 and adriamycin- novel anti-leukemia strategies. resistant75,118–120 variants ofhuman cells have been shown to exhibit defects in ceramide metabolism. In the case of adriamycin-resistant cells, ceramide is converted to a non-toxic References form (ie glucosylceramide) by glucosylceramide synthase 73 (GCS ). Introduction ofGCS into normal MCF-7 cells con- 1 Hannun YA. The sphingomyelin cycle and the second messenger 118 ferred adriamycin resistance. 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