Balance of Ceramide and Sphingosine 1 Phosphate

Comparative Biochemistry and Physiology, Part B 163 (2012) 2636
Contents lists available at SciVerse ScienceDirect
Comparative Biochemistry and Physiology, Part B

journal homepage: www.elsevier.com/locate/cbpb
Review
The control of the balance between ceramide and sphingosine-1-phosphate by

sphingosine kinase: Oxidative stress and the seesaw of cell survival and death
James R. Van Brocklyn , Joseph B. Williams
Department of Evolution, Ecology and Organismal Biology, The Ohio State University, Columbus, OH, USA
a r t i c l e
i n f o
Article history:
Received 28 February 2012
Received in revised form 9 May 2012
Accepted 12 May 2012
Available online 18 May 2012
Keywords:
Sphingolipids
Apoptosis
Autophagy
Senescence
Aging
a b s t r a c t
Sphingolipids are components of all eukaryotic cells that play important roles in a wide variety of biological
processes. Ceramides and sphingosine-1-phosphate (S1P) are signaling molecules that regulate cell fate
decisions in a wide array of species including yeast, plants, vertebrates, and invertebrates. Ceramides favor
anti-proliferative and cell death pathways such as senescence and apoptosis, whereas S1P stimulates cell proliferation and survival pathways. The control of cell fate by these two interconvertible lipids has been called
the sphingolipid rheostat or sphingolipid biostat. Sphingosine kinase, the enzyme that synthesizes S1P, is a
crucial enzyme in regulation of the balance of these sphingolipids. Sphingosine kinase has been shown to
play dynamic roles in the responses of cells to stress, leading to modulation of cell fate through a variety of
signaling pathways impinging on the processes of cell proliferation, apoptosis, autophagy and senescence.
This review summarizes the roles of sphingosine kinase signaling in these processes and the mechanisms
mediating these responses. In addition, we discuss the evidence tying sphingosine kinase-mediated stress
responses to the process of aging.
2012 Elsevier Inc. All rights reserved.
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Role of SphK in stress responses . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
SphK/S1P involvement in stress response is an evolutionarily ancient process
2.2.
Functions of SphK/S1P signaling in oxidative stress . . . . . . . . . . . . .
3.
Mechanisms by which SphK regulates cell fate . . . . . . . . . . . . . . . . . .
3.1.
Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Autophagy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Roles of S1P receptors and SphK2 . . . . . . . . . . . . . . . . . . . . .
4.
Senescence and aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.
Future directions in the comparative study of SphK in stress responses . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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26
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34
1. Introduction
Abbreviations: ABC, ATP-binding cassette; BH3, Bcl-2 homology-3; ER, endoplasmic

reticulum (ER); ERK, extracellular signal-regulated kinase; HIF, hypoxia-inducible factor;
JNK, c-jun N-terminal kinase; mTOR, mammalian target of rapamycin; N-SMase, neutral
sphingomyelinase; NF-kB, nuclear factor-kB; NLS, nuclear localization sequence; PERK,
protein kinase regulated by RNA-like ER kinase; PI3K, phosphatidylinositol-3 kinase;
ROS, reactive oxygen species; S1P, sphingosine-1-phosphate; S1PR, sphingosine-1phosphate receptor; SphK, sphingosine kinase; TNF, tumor necrosis factor; TRAF,
tumor necrosis factor receptor associated factor.
Corresponding author at: 418 Aronoff Laboratory, 300 W. 12th Ave., Columbus, OH
43210, USA. Tel.: + 1 614 292 1149; fax: + 1 614 292 2030.
E-mail address: van-brocklyn.1@osu.edu (J.R. Van Brocklyn).
1096-4959/$ see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpb.2012.05.006
Primarily lipids with a backbone of sphingosine, an 18-carbon

amino alcohol with an unsaturated hydrocarbon chain, sphingolipids
are components of all eukaryotic cell membranes, and play a wide variety of biological roles in diverse species. Much of the work aimed at
deciphering the biology of sphingolipids originally focused on complex sphingolipids such as glycosphingolipids and sphingomyelin
which have been shown to play important roles in diverse processes
including cell adhesion, membrane organization, signal transduction,
neuron myelination and bacterial toxin binding. The metabolites
of these complex sphingolipids, ceramide (Cer), sphingosine (Sph),
J.R. Van Brocklyn, J.B. Williams / Comparative Biochemistry and Physiology, Part B 163 (2012) 2636
and sphingosine-1-phosphate (S1P), were originally thought to be

merely metabolic intermediates in sphingolipid catabolism. However,
over the last two decades these lipids have been shown to have
important physiological functions, including regulation of diverse
biological processes at the cellular level such as cell proliferation,
migration and differentiation, which contribute to lymphocyte trafcking, regulation of angiogenesis, vascular development, and the
pathobiology of a number of diseases including, arteriosclerosis, asthma, autoimmune diseases, neurodegenerative diseases and cancer.
One of the most evolutionarily ancient processes regulated by
sphingolipids in eukaryotic cells is likely the regulation of cell survival
outcomes in response to cell stress agents, such as oxidative stress,
genotoxic stress, and heat stress. Whereas this has been primarily investigated in human and other mammalian cells, similar responses
have been shown in cells from a wide array of species including
yeast, plants such as Arabidopsis thaliana, non-mammalian vertebrates, such as birds and sh, and invertebrate models, including Drosophila melanogaster, the nematode Caenorhabditis elegans, and the
slime mold Dictyostelium discoideum (Oskouian and Saba, 2004).
Reactive oxygen species (ROS), such as O2, and H2O2, and OH,
are produced in the mitochondria of most all organisms as a
byproduct of normal oxidative phosphorylation. These free radicals
can damage macromolecules, including proteins, lipids and DNA
(Martindale and Holbrook, 2002), thereby imposing oxidative stress.
Although cells possess mechanisms for dealing with ROS including
anti-oxidants and repair mechanisms (Martindale and Holbrook,
2002), these defenses can be overwhelmed and oxidative stress
occurs. During periods of oxidative stress, cells can undergo
programmed cell death, or apoptosis, or they can also survive oxidative stress through other processes, including cell cycle arrest to
allow for macromolecular repair, digestion of damaged cell structures
through autophagy, or senescence. All of these processes have been
shown to be regulated by the up or down regulation of different
sphingolipids. Understanding the pathways determining in which of
these ways cells proceed in response to oxidative stress is an important challenge.
Most work on sphingolipids in stress responses has focused on the
signaling functions of two sphingolipids, ceramide and S1P. Numerous studies on invertebrates and vertebrates have shown that levels
of ceramide are increased by a variety of cell stressors, and when
ceramides increase, the cell can initiate apoptosis or programmed
cell death (Hannun and Obeid, 2008), whereas increases in S1P concentration generally promote cell survival and proliferation (Hait et
al., 2006; Hannun and Obeid, 2008). Thus the balance between the
cellular levels of these two interconvertible lipids, which has been
called the sphingolipid-rheostat (Cuvillier et al., 1996; Hait et al.,
2006), is thought to be an important mechanism controlling cell
fate (Fig. 1).
An increase in cellular concentration of ceramide can come from

several different mechanisms including activation of neutral or acid
sphingomyelinases, which remove the phosphocholine group from
sphingomyelin found in cell membranes, de novo synthesis of ceramide
occurring in the endoplasmic reticulum (ER), or from the reacylation
of sphingosine in the salvage pathway (Kitatani et al., 2008) (Fig. 2).
Through the action of ceramidases, ceramides can be converted into
sphingosine, which has been shown to have pro-apoptotic actions
similar to ceramides (Cuvillier, 2002), and sphingosine can be converted to S1P by the action of sphingosine kinases (SphK; EC 2.7.1.91).
Isoforms of sphingosine kinases (SphK), type 1 and type 2, are known
to exist, which form pro-survival S1P and, as a consequence, decrease
levels of pro-apoptotic Sph and ceramides. By determining the balance
between production of ceramides and S1P, the activity level of SphK can
be a crucial determinant of cell fate in stress responses (Fig. 1). These
pathways of sphingolipid metabolism and apoptotic versus survival
effects of sphingolipids are conserved in eukaryotic cells and have
been demonstrated to function in yeast, C. elegans, Drosophila and vertebrates (Oskouian and Saba, 2004).
How cells respond to stress agents by way of the sphingolipid
rheostat appears to be complex and incompletely understood. Here
we examine the regulation of cell fate by ceramide-S1P balance
under conditions of cell stress, with a focus on the roles of SphK
isoforms as crucial mediators of the equilibrium between ceramides
and S1P production, and ultimately of cell fate, and the similarities
and differences of these responses and the molecular mechanisms
mediating them in diverse species. The function of SphK1 is much
better understood than that of SphK2, and we therefore focus rst
on the regulation of stress responses by SphK1. However, the enzyme
SphK2 appears to have important, albeit incompletely understood,
roles in cell survival. We summarize the mechanisms used by SphK
isoforms to inuence pathways that lead to either cell survival or
death (Fig. 3). Finally, we discuss the role of SphK isoforms in regulation of cell senescence and its potential signicance to aging and variation in life span.
2. Role of SphK in stress responses
2.1. SphK/S1P involvement in stress response is an evolutionarily ancient
process
Studies on yeast cells suggest that the sphingolipid rheostat evolved
early in the history of life to regulate cell survival under conditions of
environmental stress. Yeast cells possess long chain base phosphates
dihydrosphingosine-1-phosphate and phytosphingosine-1-phosphate,
which are analogous to S1P found in other eukaryotic cells. When
yeast cells are exposed to heat shock, the viability of cells is depreciated. However, if genes for two phosphatases that dephosphorylate
O
O PO
O OH
OH OH
CH3
CH3
HN
O
OH OH
Cer-synthase
ceramide
NH2
27
CH3
SPP
Sph
ceramidase
NH 2
CH 3
S1P
SphK
Cell Proliferation
Apoptosis
Fig. 1. Diagrammatic representation of the sphingolipid rheostat. Ceramide favors a block in cell proliferation and apoptosis while S1P favors proliferation and cell survival. Note
that the rheostat is depicted as being balanced with the intermediate lipid sphingosine (Sph) on the same side as ceramide, due to the pro-apoptotic effects of sphingosine.
Thus sphingosine kinase (SphK) is in an important position to favor cell survival. SPP, sphingosine-1-phosphate phosphatase; cer-synthase, ceramide synthase.
28
Fig. 2. Sphingolipid metabolism. Production of ceramides occurs through three pathways: the sphingomyelinase pathway, the salvage pathway and de novo synthesis. Sphingosine
does not occur in de novo synthesis but only through the sphingomyelinase and salvage pathways. Sphingosine can either be phosphorylated to S1P or reacylated to ceramide.
While ceramides are constrained to membrane localization, sphingosine is not. Therefore, sphingosine can move within the cell. A major challenge facing sphingolipid research
is to determine where within the cell many of these reactions occur in response to various signals and stresses, as subcellular localization of S1P and various ceramides are likely
crucial for their biological effects. For clarity only the more important enzymes for regulation of ceramide and S1P levels are shown. SphK, sphingosine kinase; SPP, S1P phosphatase; SPL, S1P lyase; SPT, serine palmitoyl transferase; SMase, sphingomyelinase.
dihydrosphingosine-1-phosphate and phytosphingosine-1-phosphate

were deleted, hence increasing the cellular concentration of these two
molecules, survival of yeast cells during heat shock was enhanced.
(Mandala et al., 1998; Mao et al., 1999). The yeast lyase dihydrosphingosine phosphate lyase (DPL1) irreversibly degrades long chain
base phosphates to C(16) fatty aldehydes and phosphoethanolamine.
Yeast mutants that do not express DPL1, and therefore accumulate
dihydrosphingosine-1-phosphate and phytosphingosine-1-phosphate,
are also resistant to heat stress. The resistance of yeast cells to heat was
reversed by inactivation of the gene for SphK, which effectively eliminated S1P from cells (Lanterman and Saba, 1998). In addition, heat-induced
G-SH
genotoxic or
oxidative stress
ROS
ROS
N-SMase
ceramide
synthesis
p53
SphK1
ceramide
Sph
S1P
BH3 SphK2
PI-3 kinase
Bcl2
Beclin-1
mito.
Akt
p21
nucleus
cyto c
mTOR
Ca2+
apoptosis
ER
Cell cycle
arrest
autophagy
death
survival
Fig. 3. Simplied diagram depicting pathways regulating cell fate decisions involving
SphK isoforms. See text for details and references regarding each pathway. Note that
other pathways including MAP kinase isoforms, NF-kB and several ceramide-regulated
pathways have not been included for clarity. Direct targets of S1P produced by SphK
isoforms are also not shown, however, in various situations responses to S1P may be mediated by cell surface S1P receptors or through intracellular targets as discussed in the text.
cell cycle G0/G1 arrest in yeast was mediated by the sphingoid bases
dihydrosphingosine and phytosphingosine, and recovery from cell cycle
arrest was dependent on the yeast sphingosine kinases LCB4 and LCB5
(Jenkins and Hannun, 2001). Furthermore, deletion of the yeast alkaline
ceramidase gene YDC1, which cleaves dihydroceramide, increased sensitivity to heat stress (Mao et al., 2000). Interestingly, deletion of the other
yeast alkaline ceramidase YPC1, which cleaves phytoceramide, did not
affect heat stress sensitivity (Mao et al., 2000), suggesting that different
ceramide species play different roles in stress responses in yeast. Taken
together these data suggest that ceramides and long chain base phosphates are intricately involved in the process of heat resistance in yeast.
Regulation of apoptosis by ceramide and S1P is involved in the
heat stress response and cell survival in non-mammalian vertebrates.
Treatment of Japanese ounder embryos with C2-ceramide mimicked
apoptosis induced by heat stress or ultraviolet radiation (Yabu et al.,
2003). In embryonic cells of zebrash, heat stress induced apoptosis
through neutral sphingomyelinase activation and ceramide generation (Yabu et al., 2008). Interestingly, pretreatment of zebrash
cells with S1P blocks neutral sphingomyelinase-induced ceramide
generation (Yabu et al., 2008). Knockdown of neutral ceramidase in
zebrash, which would be expected to increase the ratio of ceramides
to S1P, also causes apoptosis. In birds ceramide has been shown to induce apoptosis of chicken oviduct cells (Kim et al., 2005), and hen
granulosa cells (Witty et al., 1996).
In invertebrates ceramide has been shown to be important for
apoptotic responses to radiation. Exposure of Drosophila Schneider
cells to x-ray radiation elevated ceramide levels leading to apoptosis,
and treatment with exogenous C2-ceramide mimicked apoptosis induction (Kawamura et al., 2009). Also, ceramide synthesis was necessary for radiation-induced apoptosis of C. elegans germ cells (Deng
et al., 2008). In this study, radiation caused mitochondrial accumulation of ceramide, and inhibition of ceramide accumulation by inactivation of ceramide synthase blocked radiation-induced caspase
activation and apoptosis (Deng et al., 2008). Thus, the involvement
of the sphingolipid rheostat in stress responses and cell survival is
an evolutionarily conserved mechanism in eukaryotic cells from a
wide variety of species.
2.2. Functions of SphK/S1P signaling in oxidative stress

Widely accepted by most gerontologists, (Fukagawa, 1999; Finkel
and Holbrook, 2000; Golden et al., 2002; Dufour and Larsson, 2004;
Huang and Manton, 2004), the free-radical theory of aging posits
that oxygen radicals (O2) are formed during the process of ATP formation in the mitochondria, and that the accumulation of damage
from these free radicals causes the slow deterioration of cellular processes, or aging (Gerschman et al., 1954; Harman, 1956). Free radicals
and oxidants, collectively referred to as reactive oxygen species
(ROS), are highly reactive molecules, often with an unpaired electron
in their outer shell, that readily react with lipids, proteins, and DNA
in the cell causing damage or oxidative stress. The sphingolipid rheostat plays a pivotal role in cellular responses to oxidative stress, in
that, ceramides are increased within the cell during oxidative stress.
Glutathione, an antioxidant within cells, normally binds with neutral
sphingomyelinase (N-SMase), thus inhibiting its activity. However,
when glutathione binds with ROS, N-SMase is released, and thus
sphingomyelinase becomes active converting sphingomyelin to ceramide (Liu et al., 1998). Furthermore, a sphingomyelinase inhibitor
that blocks activity of both acid and neutral sphingomyelinase
decreased ceramide generation rendering cells more resistant to oxidative stress from H2O2 (Wang et al., 2011). Thus, decreases in
ceramides appear to be associated with cell survival, whereas increases in ceramide can mediate apoptotic responses to ROS.
The enzyme SphK1 is also involved in cellular responses to ROS,
but in complex ways. Production of ROS can cause an increase or decrease in SphK1 activity, depending on the severity of oxidative damage. In this regard, oxidative stress can activate SphK1 (Ader et al.,
2008) possibly via protein kinase C (PKC)- (Jin et al., 2004). However, Ab peptide-induced toxicity in neuroblastoma cells is mediated
by ROS-induced decrease in SphK1 activity (Gomez-Brouchet et al.,
2007), and ROS can also lead to degradation of SphK1 (Pchejetski
et al., 2007). These data led to the proposal that SphK1 is a crucial
node in the response pathway to oxidative stress (Maceyka et al.,
2007), determining whether cells survive or proceed to apoptosis.
Moderate levels of oxidative stress activate SMase, but also activate
SphK1, thus shifting the ceramide-S1P balance toward S1P, suppressing apoptosis and maintaining cell survival. Excessive oxidative
stress, however, leads to SphK1 degradation, which decreases S1P,
tipping the balance of the rheostat toward apoptosis.
In addition to increasing S1P levels, SphK1 can also cause decreased ceramide levels (Maceyka et al., 2007). This may be mediated
not only by routing sphingosine away from ceramide towards S1P,
but may also involve direct feedback effects on enzymes of ceramide
synthesis. Thus, high SphK1 activity early in a stress response not only
signals for survival, but also actively decreases ceramide, implying
that degradation of SphK1 due to severe stress will not only decrease
its product S1P, but will further boost ceramide levels. This positions
SphK1 as an ideal switch enzyme to dramatically affect the ceramide/
S1P ratio and tip the balance of the sphingolipid rheostat.
The above studies suggest that SphK1 is downstream of ROS in
stress signaling, but other studies indicate that SphK1 can affect levels
of ROS. For example, SphK1 knockdown or inhibition enhanced ROS
production in several cancer cell lines, leading to increased toxicity
of doxorubicin, a chemotherapeutic agent that works through ROS
generation (Huwiler et al., 2011). In addition, SphK inhibition by
phytodimethylsphingosine leads to ROS generation, and ROS scavengers blocked phytodimethylsphingosine-induced apoptosis (Kim
et al., 2009a). There is also evidence that ROS production in conditions of stress can be stimulated by ceramide and/or sphingosine. Ceramide induces caspase 3-dependent degradation of catalase
enhancing ROS production (Iwai et al., 2003). Tumor necrosis factor
(TNF)- activated N-SMase in neurons leading to ceramide accumulation, and ceramide causes ROS generation (Barth et al., 2011). ROS
in turn led to SphK1 inhibition (Barth et al., 2011). Furthermore,
29
oxidative stress-induced apoptosis in response to paraquat required

sphingosine production by ceramidase activation and was decreased
by SphK1 activation (Abrahan et al., 2010). Thus SphK1 could block
ROS production in response to stress by decreasing the levels of
ceramide and sphingosine.
A classic example of oxidative stress, in which SphK also plays a
role, is seen in the phenomenon of ischemia-reperfusion injury.
When blood ow to cardiac myocytes is disrupted by occlusion of a
coronary artery, a series of events, largely related to energy metabolism, is set in motion that results in cell injury, and if prolonged, cell
death (Jennings et al., 1990). If reperfusion of ischemic tissue occurs
within a few minutes, free radicals are produced within these cells
and continue to be generated for hours after restoration of blood
ow (Bolli et al., 1989). However, a brief period of ischemia, prior to
an ischemia that causes injury, can protect a tissue from ischemia
reperfusion injury, a phenomenon is known as ischemic preconditioning. Ischemic preconditioning causes activation of the enzyme
SphK1, and SphK1 inhibition abolishes the protective effect of ischemic preconditioning (Jin et al., 2004). Knockout of SphK1 sensitizes
myocardial cells to reperfusion injury (Jin et al., 2007), and it has
been shown that SphK1 protects cardiac myocytes from hypoxiainduced apoptosis (Tao et al., 2007). Furthermore, pretreatment of
isolated mouse hearts with S1P also protects from ischemic injury
(Jin et al., 2002). These results indicate that the levels of S1P inuence
the outcome of ischemia, with S1P favoring cardiac myocyte survival.
Damage to DNA by external toxins, or genotoxic stress, is known
to lead to cell cycle arrest, and possibly apoptosis induced by the
tumor suppressor protein p53. This protein is also involved in cellular
responses to oxidative stress, enhancing cell survival in response to
low levels of oxidative stress, and cell death in response to severe
oxidative stress (Liu and Xu, 2011). Several recent discoveries have
revealed an important role for SphK1 downstream of p53 (Taha
et al., 2004; Heffernan-Stroud and Obeid, 2011). Whereas some chemotherapeutic agents can cause degradation of SphK1 in p53independent manner (Pchejetski et al., 2005; Bonhoure et al., 2006),
the DNA damaging agent actinomycin D was shown to lead to a
p53-dependent degradation of SphK1 protein, and SphK1 degradation was involved in the apoptotic response to DNA damage (Taha
et al., 2004). More recently, SphK1 was shown to be elevated in
knockout mice that lack p53, and to contribute to tumor formation
(Heffernan-Stroud et al., 2012). Interestingly, a double knockout
mouse that lacked p53 and SphK1 showed decreased tumor formation accompanied by an increase in cell cycle inhibitors and senescence (Heffernan-Stroud et al., 2012). These results indicate that
regulation of the sphingolipid-rheostat downstream of p53 is crucial
in determining the outcome of p53 signaling, and that SphK1 is an
important node in this pathway.
3. Mechanisms by which SphK regulates cell fate
3.1. Apoptosis
Ceramides have been known for some time to be an apoptotic
signal downstream of cell stress as well as activation of receptors
that signal apoptosis, such as fas and TNF- receptors (Hannun and
Obeid, 2008). However a seminal study in 1996 showed that
ceramide-induced apoptosis can be blocked by S1P or by SphK1 activation (Cuvillier et al., 1996). This nding was followed by numerous
studies showing that a wide variety of anti-apoptotic agents functioned to block apoptosis through activation of SphK1 (Spiegel et al.,
1998). Furthermore, overexpression of SphK1 was shown to stimulate
cell proliferation and block ceramide-induced apoptosis (Olivera et al.,
1999). These studies led to the development of the sphingolipid rheostat concept stating that the balance between the levels of ceramide
and S1P determines cell fate and that SphK is a crucial enzyme regulating that balance (Fig. 1).
30
A large number of studies regarding the effects of SphK1 on apoptosis have examined responses of cancer cells to chemotherapeutic
drugs, several of which work at least partially through induction of
oxidative stress leading to apoptosis (Maiti, 2012). A number of
chemotherapeutic drugs have also been shown to induce cancer cell
apoptosis by causing increased ceramide levels (Dimanche-Boitrel
et al., 2011). Thus, the sphingolipid rheostat concept suggests that
high SphK1 activity would tend to counteract chemotherapy because
it would promote cell survival in the face of stress caused by the toxin.
Several studies examining mechanisms of resistance to chemotherapeutic drugs have been done using the slime mold D. discoideum. A
mutagenesis screen in D. discoideum identied S1P lyase, the enzyme
that irreversibly degrades S1P, as a mediator of resistance to cisplatin
(Li et al., 2000). S1P lyase deletion in D. discoideum also increased cell
viability in stationary phase (Li et al., 2001). Further studies have
shown that in both D. discoideum and human tumor cells, that inhibition of SphK1 enhances sensitivity to chemotherapeutic drugs
(Min et al., 2004; Pchejetski et al., 2005; Sarkar et al., 2005;
Pchejetski et al., 2008; Guillermet-Guibert et al., 2009), while SphK1
overexpression has the opposite effect of making cells more resistant
to chemotherapy (Min et al., 2005; Pchejetski et al., 2005; Bonhoure
et al., 2008; Sukocheva et al., 2009).
SphK1 levels and responses of SphK1 expression and activity to
drug treatment have been shown to predict sensitivity of some cancer
cell lines to drugs (Pchejetski et al., 2005; Akao et al., 2006). Thus,
chemotherapeutic drugs decreased SphK1 activity in prostate cancer
cells which are sensitive to the drugs, while causing a modest increase in SphK1 activity in resistant cells (Pchejetski et al., 2005).
These changes were reected in the levels of the lipids in the
sphingolipid rheostat. Conditions causing SphK1 inhibition correlated
with increased ceramide/S1P ratio and enhanced apoptosis and vice
versa (Pchejetski et al., 2005). Furthermore, selection of drug resistant cells by gradual exposure to increasing drug concentrations has
in some cases been shown to be due to increased SphK1 expression.
For instance, selection of imatinib-resistant K562 cells caused
upregulation of SphK1 expression which was shown to be responsible
for the resistant phenotype (Baran et al., 2007). In addition, selection
of temozolomide-resistant glioma cells was also associated with
SphK1 upregulation (Bektas et al., 2009).
These studies on cancer cell drug sensitivity suggest that SphK1,
by controlling relative levels of S1P and ceramide, is crucial in determining cell survival under stress conditions, and that cancer cells have in
some cases taken advantage of the SphK1 pathway in order to survive
the stress of chemotherapy. In agreement, a network model of survival
signaling in T cell large granular lymphocyte leukemia showed that
SphK1 is a crucial node needed to maintain survival of these cells and
experiments with SphK inhibitors conrmed the prediction (Zhang
et al., 2008).
Several studies have examined the mechanisms by which SphK1
blocks apoptosis. The initial study regarding the anti-apoptotic
effects of SphK1/S1P, showed that S1P blocked ceramide-induced apoptosis through extracellular signal-regulated kinase (ERK) activation
and prevention of c-jun N-terminal kinase (JNK) activation (Cuvillier
et al., 1996). Signicant insight into the pathways downstream of
SphK1 also came from studies on the role of SphK1 in TNF- signaling. TNF- is well known to induce apoptosis in many cells, and one
important mechanism of this effect is sphingomyelinase activation
leading to ceramide accumulation (Obeid et al., 1993). However,
some cell types are resistant to the apoptotic effect of TNF-. SphK1
is known to be activated by TNF- signaling in such apoptosisresistant cells, and inhibition of SphK1 sensitizes cells to apoptosis
induction by TNF- (Xia et al., 1999). Prevention of TNF--induced
apoptosis appears to be caused by activation of two well-known antiapoptotic pathways downstream of SphK1, phosphatidylinositol-3
kinase (PI3K)/Akt (Osawa et al., 2001), and nuclear factor (NF)-B
(Xia et al., 2002). SphK1 directly interacts with TNF receptor associated
factor (TRAF)2 downstream of TNF- signaling to activate NF-kB (Xia

et al., 2002). More recently it was shown that S1P generated by SphK1
is necessary to act as a co-factor for TRAF2 ubiquitin ligase activity,
thus leading to I-B degradation and NF-B activation (Alvarez et al.,
2010).
These studies were signicant because they helped determine the
anti-apoptotic pathways regulated by SphK1, and they established
that a single agent can activate sphingomyelinase and SphK1, thus
leading to a dynamic regulation of sphingolipids. Prolonged TNF-
stimulation can also lead to degradation of SphK1 by cathepsin B following lysosome disruption (Taha et al., 2005). This is reminiscent of
the dual regulation of SphK1 by oxidative stress discussed above, and
indicates that the sphingolipid rheostat is a dynamic process, and
duration or intensity of a signal or a stress can affect the balance of
the rheostat, to a large degree by regulation of SphK1, tipping it in
favor of apoptosis with increased time or severity.
Another mechanism of apoptosis regulation by SphK/S1P is through
members of the Bcl-2 family of apoptosis-regulating proteins. S1P
is known to block apoptosis through inhibition of events which are
downstream of Bcl-2 family proteins, including cytochrome c and
Smac/DIABLO release from mitochondria (Cuvillier and Levade, 2001).
In agreement, SphK/S1P signaling has been shown to lead to accumulation of the anti-apoptotic protein Bcl-2. Thus, moderate SphK1 overexpression upregulated Bcl-2 as well as activating PI3K (Limaye et al.,
2005). Further evidence comes from experiments overexpressing
ceramidase, the enzyme that deacylates ceramide to sphingosine.
Ceramidase overexpression has been shown to upregulate Bcl-2 expression via production of S1P (Wang et al., 2004). However, another study
found that SphK1 overexpression failed to upregulate Bcl-2, and instead
Bcl-2 overexpression upregulated SphK1 (Bektas et al., 2005). Thus,
SphK1 may be either upstream or downstream of Bcl-2, perhaps
depending upon the cells examined.
Other Bcl-2 family members besides Bcl-2 itself are also regulated
by SphK/S1P signaling. SphK1 overexpression blocks apoptosis induced by the chemotherapeutic drug imatinib in chronic myelogenous leukemia cells by preventing the decrease in expression of the
anti-apoptotic Bcl-2 family proteins Bcl-xl and MCL-1, while decreasing imatinib-induced upregulation of pro-apoptotic Bim (Bonhoure
et al., 2008). A recent study showed that SphK1-induced downregulation of Bim in glioma cells is mediated through activation of
PI3K/Akt/FOXO3a signaling (Guan et al., 2011). Thus, SphK1 appears
to modulate levels of several members of the Bcl-2 family in a direction favoring cell survival.
Finally, SphK1 could modulate apoptotic signaling, not only
through production of S1P, but also through decreasing the levels of
pro-apoptotic ceramide and sphingosine (Kohama et al., 1998). This
may be mediated by routing sphingolipids through S1P to irreversible
degradation by S1P lyase (Maceyka et al., 2005). However, SphK1
may also affect synthesis of ceramide by the de novo and salvage
pathways. In this regard, SphK1 overexpression decreases incorporation of [ 3 H]serine or [ 3 H]palmitate, the precursors in the synthesis of
sphingolipids in the endoplasmic reticulum, into ceramides (Maceyka
et al., 2005), and SphK1 knock down by siRNA increases de novo synthesis of ceramide, particularly in the mitochondria-enriched fraction,
leading to apoptosis through Bax oligomerization and cytochrome c
release (Taha et al., 2006). Thus, S1P could act as a negative feedback
regulator of sphingolipid biosynthesis, and it has been proposed
that S1P may inhibit serine palmitoyltransferase and/or ceramide
synthase (van Echten-Deckert et al., 1997; Spiegel and Milstien,
2003) (Fig. 2).
3.2. Autophagy
Whereas most studies on the role of SphK in cell survival have
focused on regulation of apoptotic responses, several recent studies
have shown that pathways for autophagy, a catabolic process that
involves the degradation of cellular components through the formation of autophagosomes that fuse with lysosomes, are also regulated
by sphingolipids. A process utilized by cells under conditions of nutrient depletion or other stress (Maiuri et al., 2007), autophagy is often
a survival pathway for cells by elimination of damaged organelles,
however when stress becomes extreme, autophagy can also lead
to cell death (Shintani and Klionsky, 2004). Autophagic cell death is
sometimes referred to as Type II cell death, whereas apoptosis is
referred to as Type I cell death.
Although an increase in ceramide almost always favors apoptosis
and an increase in S1P blocks it, regulation of autophagy by sphingolipids is more complex, as both ceramide and S1P have been shown
to stimulate autophagy. Ceramide induces autophagy via upregulation
of Beclin-1 (Scarlatti et al., 2004), a well-known autophagy inducing
protein, and upregulation of BNIP-3 (Daido et al., 2004) usually resulting
in autophagic cell death. SphK1/S1P-induced autophagy appears to be
mediated by different pathways and is generally a survival response.
For example, SphK1 overexpression induced autophagy in MCF-7 cells
(Lavieu et al., 2006), and nutrient starvation of MCF-7 cells led to
SphK1 activation and autophagy. SphK1 inhibition blocked starvationinduced autophagy leading to apoptosis, demonstrating that SphK1induced autophagy was important for cell survival in starvation conditions (Lavieu et al., 2006). Interestingly, yeast SphK enzymes are also
activated by nutrient deprivation (Lanterman and Saba, 1998), again
illustrating the conserved nature of SphK as a response to stress.
Direct comparisons of the signaling pathways mediating ceramide
and S1P-induced autophagy show different mechanisms. Ceramideinduced autophagy of MCF-7 cells was mediated by inhibition of
PI3K/Akt signaling and a strong upregulation of Beclin-1. In contrast,
SphK1-induced autophagy was mediated by decreased activity of
mammalian target of rapamycin (mTOR) with no effect on PI3K/Akt.
Furthermore, SphK1 caused a more modest increase in Beclin 1 than
did ceramide (Lavieu et al., 2006). These differences between ceramide and SphK1-induced autophagy signaling may be crucial in determining whether autophagy is a survival or death response. The
lack of inhibition of PI3K/Akt in SphK1-induced autophagy is in keeping with the well-known anti-apoptotic effect of Akt. Furthermore,
the balance of Beclin-1 and Bcl-2 may be important in determining
outcome (Lavieu et al., 2007), as Bcl-2 has been shown to interact
with Beclin-1 to inhibit autophagy, and the Beclin-1/Bcl-2 balance
has been suggested to maintain moderate levels of autophagy compatible with cell survival (Pattingre et al., 2005). It is interesting to
note that SphK1 can upregulate Bcl-2 (Limaye et al., 2005). Thus, by
causing only a modest increase in Beclin-1 and an increase in Bcl-2,
SphK1 favors the survival side of autophagy. Bcl-2 overexpression
can also induce increased SphK1 expression (Bektas et al., 2005),
which may suggest a complex interplay between the ceramide-S1P
and Beclin-1/Bcl-2 balance. Based on this interplay of SphK1regulated autophagic and apoptotic pathways it has been suggested
that autophagy may be the key mechanism by which the sphingolipid
rheostat controls cell survival (Lavieu et al., 2007).
Knockdown of sphingosine-1-phosphohydrolase-1 (SPP1), a specic phosphatase that degrades S1P, also induced autophagy, however, in contrast to SphK1 overexpression, this response did not involve
effects on Beclin-1 or mTOR, but rather was mediated by activation of
ER stress and the unfolded protein response (Lpine et al., 2011).
Nevertheless, in this case, the autophagy induced also led to cell
survival rather than death, indicating that S1P may induce autophagy
by varying mechanisms depending upon how or where in the cell it
is produced, however S1P-induced autophagy is consistently a survival response. One pathway that does appear to be consistently involved in S1P-induced survival via autophagy is Akt. Knockdown of
sphingosine-1-phosphohydrolase-1 led to increased phospho-Akt
via activation of the unfolded protein response sensor PERK, and
PI3K (Lpine et al., 2011). Thus, Akt may be an important determinant
of cell survival within S1P-induced autophagy.
31
3.3. Roles of S1P receptors and SphK2

In cells from vertebrate animals, S1P can regulate cellular processes
by two separate methods, by binding to ve specic G protein-coupled
cell surface receptors termed S1PR15, or independently of receptors
through several intracellular targets (Strub et al., 2010), which are less
well understood. There is clear evidence that many SphK-regulated
responses are mediated independently of S1P receptors. One crucial
nding is the existence of sphingolipid rheostat regulation of cell
survival in invertebrate organisms such as Drosophila, nematodes and
yeast, as these organisms do not possess S1P receptors. Thus, the evolutionarily oldest pathways of sphingolipid-mediated stress response
regulation must be receptor-independent. Furthermore, SphK1 overexpression in embryonic mouse broblasts devoid of expression of
S1P receptors still promotes growth and survival (Olivera et al., 2003),
and therefore, these receptor-independent pathways still exist in
mammalian cells.
Nevertheless, many of the actions of S1P on vertebrate cells have
been shown to be mediated by S1P receptors. Clearly S1P signaling
through S1P receptors can activate anti-apoptotic pathways, such as
PI-3 kinase/Akt and ERK MAP kinases (Young and Van Brocklyn,
2006). Furthermore, transporters of the ATP-binding cassette (ABC)
family, which are known to export S1P extracellularly (Kim et al.,
2009b), have in some cases been shown be necessary for S1Pmediated survival responses (Nieuwenhuis et al., 2009). Therefore a
clear difference between invertebrate and vertebrate animals with regard to the role of S1P in stress responses is that both receptorindependent and receptor-mediated responses exist in vertebrates,
while S1P responses in invertebrates are solely receptorindependent.
One of the best studied S1P receptor-mediated survival response involves the role of SphK/S1P signaling in protection from ischemiareperfusion injury. Protection of cardiomyocytes from hypoxia-induced
apoptosis by SphK1 involved ERK activation via S1PR1 (Tao et al.,
2009). S1PR1 signaling was also shown to protect against ischemia
reperfusion injury in kidney (Bajwa et al., 2010). However, the function
of S1P receptors in ROS mediated injury is complex, as S1PR2 signaling in
renal tubule cells decreases SphK1 expression, exacerbating ischemia
reperfusion injury (Won Park et al., 2012). Conversely, antagonism of
the receptor S1PR2 protects kidneys from ischemia-reperfusion injury
and blocks H2O2-induced necrosis via increased SphK1 expression
(Won Park et al., 2012). Furthermore, while S1PR3 can be antiapoptotic in some cases (Nieuwenhuis et al., 2009), it has also shown
to increase ROS leading to cardiac brosis in SphK1 transgenic mice
(Takuwa et al., 2010).
Autophagy induced by downregulation of sphingosine-1phosphohydrolase-1 has been shown not to be receptor mediated
(Lpine et al., 2011), but it has been suggested that S1P receptor signaling can induce autophagy. Thus, knockdown of S1PR5 by siRNA
blocked autophagy induced by extracellular S1P in prostate cancer
cells (Chang et al., 2009). However, high concentrations of S1P,
5 mM, were required to induce autophagy in this system, far beyond
the concentration of S1P needed to stimulate S1PR5, as the dissociation constant of S1PR5 for S1P is approximately 10 nM (Malek et al.,
2001). High concentrations of exogenous S1P are sometimes used to
investigate potential intracellular effects, as S1P is poorly taken up
by cells. Thus, it is unclear in this instance if S1P-induced autophagy
was entirely receptor-driven or at least partially intracellularly
mediated.
It is thought that the effects of SphK1 are more likely mediated by
S1P receptor-dependent signaling than SphK2-dependent effects.
SphK1 is known to translocate to the plasma membrane upon activation (Pitson et al., 2003), and this translocation is necessary for transforming ability of SphK1 (Pitson et al., 2005). However, SphK2 has
been shown to located in the nucleus and the cytoplasm, and to
sometimes associate with the ER (Wattenberg, 2010), suggesting
32
the effects of S1P produced by SphK2 may be mediated intracellularly.

Furthermore, overexpression of SphK1, but not SphK2, leads to
enhanced S1P release through ABC proteins (Takabe et al., 2010).
Nevertheless SphK2 has important roles in cell survival responses,
and it can associate with plasma membrane (Maceyka et al., 2005)
and lead to export of S1P (Schnitzer et al., 2009).
Overexpression of SphK2, which contains a Bcl-2 homology-3
(BH3) domain, was initially shown to be pro-apoptotic (Liu et al.,
2003). SphK2 interacts with Bcl-Xl through its BH3 domain, and
mutation of the BH3 domain reduced but did not eliminate its proapoptotic activity (Liu et al., 2003). Mutation of the ATP-binding
sequence to eliminate the enzymatic sphingosine kinase activity
also reduced the pro-apoptotic activity of SphK2 when it was overexpressed (Maceyka et al., 2005). Combining both mutations eliminated the apoptotic activity demonstrating that the BH3 domain and SphK
activity are necessary for the full apoptotic response (Maceyka et al.,
2005).
In agreement with a pro-apoptotic role for SphK2, in contrast to
the anti-apoptotic role of SphK1, SphK2 expression was shown to
increase sensitivity to chemotherapeutic drugs while SphK1 expression decreased sensitivity (Min et al., 2007). However, results of
experiments knocking down SphK2 expression provided contrasting
results. SphK2 knockdown was shown to decrease proliferation and
enhance apoptosis more potently than SphK1 knockdown in glioblastoma cells (Van Brocklyn et al., 2005) and several other cancer cell
types (Gao and Smith, 2011), and SphK2 inhibition increased apoptosis of therapy-resistant breast cancer cells by blocking NF-kB survival
signaling (Antoon et al., 2011). In addition, SphK2 inhibition induced
autophagy of several different types of carcinoma cells, leading to
autophagic cell death (Beljanski et al., 2010). Thus, SphK2 appears
to have toxic as well as survival effects.
One possible explanation for the pro-survival effects of SphK2 was
that SphK2 enhances expression of the cyclin-dependent kinase inhibitor p21 in response to the chemotherapeutic drug doxorubicin.
Knocking down SphK2 blocks p21 induction, decreases G2/M arrest,
and enhances doxorubicin-induced apoptosis (Sankala et al., 2007).
It was suggested that SphK2 is crucial for the activation of the G2/M
cell cycle checkpoint through p21 induction, thus allowing for repair
of DNA damage and preventing apoptosis (Sankala et al., 2007). In
agreement, pharmacological inhibition or siRNA-mediated knockdown of either SphK1 or SphK2 enhanced chemotherapeutic druginduced ceramide generation and apoptosis (Nemoto et al., 2009),
and SphK2 knockdown sensitized colon cancer cells to sodium
butyrate-induced apoptosis (Xiao et al., 2012). Furthermore, hypoxia
of A549 lung cancer cells was shown to induce increased SphK2
expression and S1P release, which led to protection from apoptosis
through autocrine/paracrine signaling via S1PR1/3 (Schnitzer et al.,
2009). Nevertheless, the pro-apoptotic functions ascribed to SphK2
are not merely artifacts of overexpression experiments, as renal
mesangial cells from SphK2 / mice were resistant to staurosporineinduced apoptosis. This correlated with upregulation of the antiapoptotic Bcl-Xl and a lack of decreased Akt activation in response to
staurosporine in these cells (Hofmann et al., 2008). SphK2 may play
important roles in checkpoints that are necessary for cells to survive
stress, but may also be able to induce apoptosis, perhaps in conditions
of severe stress.
Since both SphK1 and SphK2 produce the same product, S1P, it is
intriguing that they can have different biological effects; however
the particular role played by S1P may depend upon the subcellular
localization of its production. Whereas SphK1 is predominantly cytoplasmic and translocates to the plasma membrane upon activation,
SphK2 has been shown to localize to the cytoplasm and nucleus
(Wattenberg, 2010). SphK2 has a nuclear localization sequence
(NLS) (Igarashi et al., 2003), and nuclear localization has often been
associated with its antiproliferative effects. Mutation of the NLS
prevented SphK2 from localizing to the nucleus and also prevented
its antiproliferative effects (Igarashi et al., 2003). Furthermore,

targeting SphK1 to the nucleus as a hybrid with the SphK2 NLS
renders it inhibitory to proliferation (Igarashi et al., 2003). Another
study showed that serum deprivation caused SphK2 to translocate
to the nucleus, thus leading to inhibition of proliferation (Okada et
al., 2005). The antiproliferative effect of nuclear SphK2 is likely mediated by its interaction with histone H3 and role in histone acetylation
(Hait et al., 2009). S1P produced by SphK2 in association with histone
H3 inhibits histone deacetylase, leading to enhanced histone acetylation and transcription of the cyclin-dependent kinase inhibitor p21
(Hait et al., 2009).
SphK2 has roles outside the nucleus. Protein kinase D phosphorylates SphK2, leading to its export from the nucleus (Ding et al., 2007),
indicating that the subcellular localization of SphK2 can be regulated
by signaling pathways. Furthermore, SphK2 translocated to ER during
serum starvation and ER localization of S1P production rendered it
pro-apoptotic by releasing Ca 2 + that was needed for cytochrome c
release from mitochondria and by increasing ceramide biosynthesis,
probably through the salvage pathway (Maceyka et al., 2005). In
agreement with the concept that S1P accumulation in ER is toxic,
S1P production by SphK2, but not by SphK1, was toxic to cerebellar
granule cells from S1P lyase-decient mice (Hagen et al., 2009). The
authors proposed that addition of S1P to these cells led to its uptake,
dephosphorylation, and rephosphorylation by SphK2 in the ER where
its accumulation, because of a lack of lyase in these cells, rendered it
apoptotic. In contrast sphingosine added to these cells was phosphorylated to S1P by SphK1 either in the cytosol or at the plasma membrane and did not cause apoptosis.
SphK2 has also been shown to play a role in ischemic preconditioning. Hypoxic preconditioning increased SphK2 expression in
cerebral vascular endothelial cells (Wacker et al., 2009), and SphK2
knockout mice had larger ischemic lesions and more severe neurologic
decits in response to cerebral ischemia (Pfeilschifter et al., 2011).
SphK2 knockout mice are not protected from ischemic brain injury by
ischemic preconditioning and lose HIF-1 upregulation (Yung et al.,
2011). SphK2 also contributes to reduced ischemia reperfusion injury
in kidneys (Jo et al., 2009).
4. Senescence and aging

A recent study showed that senescent cells are important mediators of aging (Baker et al., 2011). In addition to the cellular processes
of apoptosis and autophagy, ceramides have also been implicated in
cell senescence (Saddoughi et al., 2008). Ceramide levels increase as
broblasts enter senescence, and ceramide mediates changes in expression of cell cycle-related proteins associated with senescence (Venable
et al., 1995). As animals age, their telomeres, regions of repetitive nucleotide sequences at the end of chromosomes, tend to shorten (Aubert
and Lansdorp, 2008). Ceramides within the cell can mediate shortening
of telomeres by multiple mechanisms (Saddoughi et al., 2008). Ceramide has also been directly associated with aging, based upon the
fact that ceramide synthase genes are homologues of yeast longevity assurance genes (LAG), that are known to regulate yeast lifespan (Obeid
and Hannun, 2003).
Few studies have examined the roles of SphK/S1P signaling in
senescence, however the interconnected nature of the sphingolipid
rheostat and the effect of SphK on ceramide levels, supports the
idea that S1P is likely important in this process. Treatment of endothelial cells with glycated collagen led to elevated ceramide levels and
autophagy and eventually to cell senescence (Patschan et al., 2008).
Inhibition of autophagy decreased senescence but increased apoptosis,
and the authors thus suggested that autophagy acts a switch between
apoptosis and senescence (Patschan et al., 2008). At the later stages of
glycated collagen treatment they saw an increase in S1P, suggesting
that S1P's anti-apoptotic effects, and possibly pro-autophagic effects,
could modulate the development of senescence (Patschan et al., 2008),

but exactly how S1P functioned in this system was not clear.
Degradation of SphK1 mediated by p53 protein leads to enhanced
tumor cell senescence (Heffernan-Stroud et al., 2012), suggesting that
the role of SphK1 degradation in p53-stimulated senescence was to
lead to increased ceramide and sphingosine. Although undoubtedly
true, it does not preclude an active role for S1P in regulating senescence. For example, senescent endothelial cells have been shown to
have increased expression of S1PR2 (Gruber et al., 2010). Increased
S1PR2 signaling in these cells led to enhanced activation of PTEN
and inhibition of Rac, which was involved in impairment of function
in senescent endothelial cells (Estrada et al., 2008). This may seem
counterintuitive since S1P receptors can stimulate cell proliferation
and survival pathways. However, S1PR2 often appears to function in
opposition to the other two commonly expressed receptors, S1PR1
and S1PR3, which have been associated with such pathways. Therefore, in order to understand the contribution of the SphK/S1P side of
the rheostat to senescence, it will likely be important to understand
not only changes in lipid metabolism but in the pathways mediating
the downstream effects of S1P.
Given that autophagy decreases with age, it may be that decreased
levels of autophagy contribute to senescence and aging (Donati,
2006). Since SphK1 stimulates autophagy as a survival response,
this suggests that either SphK activity or cellular responses to increases
in S1P may change with age, contributing to a decrease in the ability of
cells to successfully deal with stress through survival autophagy, and
hence enhanced cell death or senescence. Little is known regarding
the changes in SphK isoforms with age, however, one study found the
SphK2 expression decreased in hearts from aging mice (Vessey et al.,
2009). This reduction in expression of SphK2 was correlated with a
reduced efciency of ischemic preconditioning in older mice. SphK2
knockout has been shown to increase ischemia reperfusion injury in
mouse hearts (Vessey et al., 2011). Together these data suggest that
SphK2 may be important for mediating resistance to oxidative stress,
and that decreased SphK2 with aging could render tissues more susceptible. It is also possible that decreased SphK2 in mitochondria with age
could lead to aberrant mitochondrial respiration causing increased
ROS production, as proper assembly of complex IV requires SphK2
(Strub et al., 2011).
Recently it was shown that decreasing sphingolipid synthesis,
through either pharmacological or genetic blockage of serine
palmitoyltransferase, the enzyme that mediates the rst step in production of sphingolipids, increased lifespan in yeast (Huang et al.,
2012). This increase in longevity was facilitated by decreased activity
of the Sch9 protein kinase, a homolog of mammalian ribosomal S6
kinase which is activated downstream of TOR1. Decreasing yeast
sphingolipid synthesis also enhanced resistance to heat and oxidative
stress (Huang et al., 2012). Decreased levels of several sphingolipids,
including long chain bases and long chain base phosphates as well as
more complex sphingolipids, were seen in these experiments, and
therefore it was not clear which sphingolipids were responsible for
increased stress resistance and lifespan.
Whereas little is known regarding the potential role of S1P in invertebrate aging, ceramides have been linked to regulation of lifespan
in yeast and Drosophila, but surprisingly several studies have shown
that ceramides increase longevity in these species. Overexpression
of the yeast alkaline ceramidase YDC1 led to increased sensitivity to
oxidative stress, dysfunction and fragmentation of mitochondria,
increased levels of ROS, apoptosis and reduced chronological lifespan
(Aerts et al., 2008). These effects of YDC1 overexpression were
reversed by addition of C6-dihydroceramide, and the authors suggested that since changes in mitochondrial morphology and function
occurred rst that these effects may be due to ceramide-induced
changes in mitochondrial membrane stability (Aerts et al., 2008).
Similarly, inactivating mutation of alkaline ceramidase in Drosophila
increases ceramide levels and lifespan (Yang et al., 2011). This was
33
associated with lower mitochondrial ROS production, greater resistance to oxidative stress and slower decline in ATP levels with age,
suggesting a preservation of mitochondrial function (Yang et al.,
2011). Mutation of Drosophila ceramide transfer protein, which transfers ceramide from the ER to the Golgi, decreases sphingomyelin and
ceramide, and this leads to increased membrane uidity, causing cells
to be more susceptible to oxidative stress, and decreasing lifespan
(Rao et al., 2007). Thus, in addition to the pathways of stress response
signaling, changes in membrane composition due to alterations in
sphingolipids could affect stress resistance and longevity. It remains
to be determined whether this role of ceramides in aging is also applicable to vertebrates.
Another intriguing possible link of the sphingolipid rheostat to
aging can be seen in the involvement of SphK in oxidative stress
signaling. Originally proposed over half a century ago and now regarded
as the most plausible, the free radical theory of aging states that cumulative damage from oxidative stress leads to progressive cellular and
tissue malfunction and is the root cause of aging (Harman, 1956). However some recent studies indicate that, whereas high levels of oxidative
stress are detrimental, low levels can extend lifespan (Ristow and
Schmeisser, 2011). This observation has been explained based on the
concept of hormesis, the idea that low levels of stress can be benecial
by enhancing stress resistance (Gems and Partridge, 2008). For example, extension of lifespan in C. elegans by caloric restriction was mediated by increased levels of ROS and was blocked by antioxidants
(Schulz et al., 2007). Other studies have shown that increased resistance
to oxidative stress in response to low levels of ROS occurs not only in
nematodes but also in rodents and humans (Ristow and Schmeisser,
2011). Inhibition of respiration in C. elegans extends life span by production of ROS that in turn leads to HIF-1 activation (Lee et al., 2010). SphK1
can mediate HIF-1 induction in response to ROS (Ader et al., 2008). In
addition, SphK1 activation by low levels of oxidative stress and degradation in response to high levels is reminiscent of the concept of
hormesis. These data raise the intriguing possibility that low levels of
oxidative stress may extend life span through SphK1-mediated modulation of the sphingolipid rheostat, thus affecting survival versus death
pathways such as autophagy, senescence and apoptosis.
5. Future directions in the comparative study of SphK in
stress responses
Many of the studies discussed in this review clearly demonstrate
the evolutionarily conserved importance of SphK/S1P signaling in
stress responses, however, little is known regarding the variation in
how these pathways function among different taxa. No studies have
directly compared the functioning of these pathways across diverse
species in order to understand whether organisms have evolved differences in how these pathways function, and whether such differences could contribute to the variation seen in stress resistance, and
possibly lifespan, between species. Furthermore, the use of laboratory
strains of model organisms, while useful for molecular work, prevents the examination of how natural selective pressures may have
modied the functions of sphingolipid pathways in stress resistance.
Comparative studies examining these pathways in animals captured
in the wild, therefore, hold enormous potential to illuminate the
role and importance of these pathways in stress resistance in animals
exposed to selective pressures of their natural environments.
Acknowledgments
We thank members of the Williams lab group for critically reading
the manuscript and making valuable comments. The coauthors of this
paper disagreed about usage of the term sphingolipid rheostat as a
concept that describes the balance between production of S1P and
ceramides. The term sphingolipid biostat was considered a viable
alternative. Because a rheostat is a variable resistor for regulating a
34
current, the term sphingolipids rheostat did not precisely describe

what we intend to convey about this mechanism that achieves balance between S1P and ceramide production. Yet, because this nomenclature seems to be rmly ensconced in the literature, we have used
the term in this paper, despite its ambiguity to those outside of the
coterie that works on S1P-ceramide signaling. We hope that this discussion will stimulate others to begin a dialogue about the appropriate nomenclature for this important mechanism.
Funding for this project came from the National Science Foundation
# IOS-1036914.
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Comparative Biochemistry and Physiology, Part B 163 (2012) 2636

Contents lists available at SciVerse ScienceDirect

Comparative Biochemistry and Physiology, Part B

The control of the balance between ceramide and sphingosine-1-phosphate by

Abbreviations: ABC, ATP-binding cassette; BH3, Bcl-2 homology-3; ER, endoplasmic

Primarily lipids with a backbone of sphingosine, an 18-carbon

and sphingosine-1-phosphate (S1P), were originally thought to be

An increase in cellular concentration of ceramide can come from

dihydrosphingosine-1-phosphate and phytosphingosine-1-phosphate

2.2. Functions of SphK/S1P signaling in oxidative stress

oxidative stress-induced apoptosis in response to paraquat required

factor (TRAF)2 downstream of TNF- signaling to activate NF-kB (Xia

3.3. Roles of S1P receptors and SphK2

the effects of S1P produced by SphK2 may be mediated intracellularly.

its antiproliferative effects (Igarashi et al., 2003). Furthermore,

4. Senescence and aging

could modulate the development of senescence (Patschan et al., 2008),

current, the term sphingolipids rheostat did not precisely describe

Dimanche-Boitrel, M.T., Rebillard, A., Gulbins, E., 2011. Ceramide in chemotherapy of

Spiegel, S., Milstien, S., 2003. Sphingosine-1-phosphate: an enigmatic signalling lipid.

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