Nitric Oxide Donors Induce Stress Signaling via Ceramide
Formation in Rat Renal Mesangial Cells*
Andrea
Huwiler
§¶,
Josef
Pfeilschifter§, and
Henk
van den
Bosch
From the
Center for Biomembranes and Lipid
Enzymology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The
Netherlands and § Zentrum der Pharmakologie, Klinikum der
Johann Wolfgang Goethe-Universität, D-60590 Frankfurt am Main,
Germany
 |
ABSTRACT |
Exogenous NO is able to trigger
apoptosis of renal mesangial cells, and thus may contribute to
acute lytic phases as well as to resolution of glomerulonephritis.
However, the mechanism involved in these events is still unclear. We
report here that chronic exposure of renal mesangial cells for 24 h to compounds releasing NO, including spermine-NO,
(Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate (MAHMA-NO), S-nitrosoglutathione (GS-NO), and
S-nitroso-N-acetyl-D,L-penicillamine (SNAP) results in a potent and dose-dependent increase in
the lipid signaling molecule ceramide. Time courses reveal that
significant effects occur after 2-4 h of stimulation with NO donors
and reach maximal levels after 24 h of stimulation. No acute
(within minutes) ceramide production can be detected. When cells were
stimulated with NO donors in the presence of phorbol ester, a direct
activator of protein kinase C, both ceramide production and DNA
fragmentation are completely abolished. Furthermore, addition of
exogenous ceramide partially reversed the inhibitory effect of phorbol
ester on apoptosis, thus suggesting a negative regulation of protein
kinase C on ceramide formation and apoptosis. In contrast to exogenous
NO, tumor necrosis factor (TNF)-
stimulates a very rapid and
transient increase in ceramide levels within minutes but fails to
induce the late-phase ceramide formation. Moreover, TNF fails to
induce apoptosis in mesangial cells.
Interestingly, NO and TNF
cause a chronic activation of acidic and
neutral sphingomyelinases, the ceramide-generating enzymes, whereas
acidic and neutral ceramidases, the ceramide-metabolizing enzymes, are
inhibited by NO, but potently stimulated by TNF
. Furthermore, in the
presence of an acidic ceramidase inhibitor, N-oleoylethanolamine, TNF
leads to a sustained
accumulation of ceramide and in parallel induces DNA fragmentation.
In summary, our data demonstrate that exogenous NO causes a chronic
up-regulation of ceramide levels in mesangial cells by activating
sphingomyelinases and concomitantly inhibiting ceramidases, and that
particularly the late-phase of ceramide generation may be responsible
for the further processing of a proapoptotic signal.
 |
INTRODUCTION |
In recent years, nitric oxide
(NO),1 a gas previously
considered a potentially toxic chemical, has become established as a diffusible universal messenger mediating cell-cell communication throughout the body. NO is a well known mediator of blood vessel relaxation that helps to maintain blood pressure. In the central nervous system NO acts as a nonconventional type of neurotransmitter contributing to long term potentiation. In addition NO is responsible for parts of the cytotoxic activity of macrophages that helps to defeat
microbes and tumor cells. Excessive and uncontrolled production of NO
is associated with severe diseases like septic shock, stroke,
neurodegeneration, diabetes mellitus, arthritis, and other forms of
acute and chronic inflammation (1-4). In mammals, the synthesis of NO
is catalyzed by nitric-oxide synthase (NOS), which exists in several
isoforms. NOS catalyzes the oxidation of L-arginine to
citrulline and NO.
Renal mesangial cells exposed to proinflammatory cytokines like
interleukin-1 or tumor necrosis factor-
express an inducible NOS and
produce large amounts of NO (5, 6), which may contribute to certain
forms of glomerulonephritis (4, 7, 8). Glomerular mesangial and
endothelial cells are not only production sites of NO but are also
themselves targets for NO and undergo apoptotic cell death upon
exposure to high concentrations of NO (9, 10). Apoptosis is a
controlled biological strategy to remove damaged or unwanted cells from
a given tissue and thus is involved in important physiological and
pathophysiological processes (11, 12).
NO-induced apoptosis has been described for a variety of cell types
including macrophages (13, 14), neurons (15, 16), thymocytes (17, 18),
and glomerular mesangial, endothelial, and epithelial cells (9, 10). By
contrast, under certain circumstances NO can inhibit apoptosis for
example in endothelial cells where NO inhibits interleukin
1
-converting enzyme-like proteases (19).
In this context, another molecule, the sphingolipid ceramide, has
attracted attention as a candidate regulator of apoptosis. Ceramide is
generated by sphingomyelinase-catalyzed sphingolipid turnover and has
been characterized as an important intracellular mediator of stress
signaling, which under certain conditions can cause apoptosis (20,
21).
In this study we present evidence that exogenously applied NO induces a
chronic up-regulation of intracellular ceramide levels and that this
delayed increase in ceramide is paralleled by the onset of apoptosis.
 |
EXPERIMENTAL PROCEDURES |
Chemicals--
(Z)-1-{N-Methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate
(MAHMA-NO),
(Z)-1-{N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]amino}-diazen-1-ium-1,2-diolate (spermine-NO), S-nitrosoglutathione (GS-NO), and
S-nitroso-N-acetyl-D,L-penicillamine (SNAP) were from Alexis Corp., Läufelfingen, Switzerland;
[14C]serine (specific activity, 53 Ci/mol) and
[14C]sphingomyelin (specific activity, 55 Ci/mol) were
from Amersham Pharmacia Biotech Buckinghamshire, UK;
[14C]ceramide (specific activity 55 Ci/mol) was from ICN
Pharmaceuticals, Inc., Irvine, CA;
12-O-tetradecanoylphorbol-13-acetate (TPA) was purchased
from Calbiochem, Lucerne, Switzerland; dibutyryl cyclic GMP and
N-oleoylethanolamine were from Sigma; all cell culture nutrients were from Life Technologies, Inc., Breda, the Netherlands; TNF
was a gift of Knoll AG, Ludwigshafen, Germany.
Cell Culture--
Rat renal mesangial cells were cultivated and
characterized as described previously (22, 23). In a second step,
single cells were cloned by limited dilution on 96-well plates. Clones with apparent mesangial cell morphology were characterized by positive
staining for the intermediate filaments desmin and vimentin, which is
considered to be specific for myogenic cells, positive staining for Thy
1.1 antigen, and negative staining for Factor VIII-related antigen and
cytokeratin, excluding endothelial and epithelial contaminations,
respectively. For the experiments in this study passages 10-23 were used.
Lipid Extraction and Ceramide Quantitation--
Confluent
mesangial cells in 30-mm diameter dishes were labeled for 24 h
with [14C]serine (0.2 µCi/ml) and stimulated as
indicated. The reaction was stopped by extraction of lipids (24), and
ceramide was resolved by sequential one-dimensional TLC using
chloroform/methanol/ammonia (65:35:7.5; v/v) followed by
chloroform/methanol/acetic acid (9:1:1; v/v). Spots corresponding to
ceramide were analyzed and quantitated using a Berthold (Nashua, NH) LB
2842 automatic TLC scanner.
Apoptosis Assay--
Confluent mesangial cells in 60-mm diameter
dishes were incubated with the indicated stimuli in DMEM containing 0.1 mg/ml fatty acid-free bovine serum albumin for the indicated time
periods. Thereafter, oligonucleosomal DNA fragmentation, a
characteristic biochemical feature of apoptotic cell death, was
measured using a nucleosome DNA-enzyme-linked immunosorbent assay
(Boehringer), which quantitatively records histone-associated DNA fragments.
Acidic and Neutral Sphingomyelinase Assay--
Confluent
mesangial cells in 60-mm diameter dishes were incubated with the
indicated stimuli in DMEM containing 0.1 mg/ml fatty acid-free bovine
serum albumin for the indicated time periods. Thereafter cells were
homogenized in lysis buffer containing 50 mM sodium
acetate, pH 4.5, 0.5% Triton X-100, 5 mM
MgCl2, 1 mM EDTA for the acidic
sphingomyelinase, and 50 mM Tris, pH 7.4, 0.5% Triton
X-100, 5 mM MgCl2, 1 mM EDTA, 5 mM dithiothreitol for the neutral sphingomyelinase. Cell
homogenates were centrifuged for 10 min at 14,000 × g,
and the supernatant was taken for an in vitro assay. 100 µg of protein in a total volume of 100 µl was incubated for 30 min
at 37 °C with 20 nCi of [14C]sphingomyelin.
Thereafter, the reaction was stopped by the addition of 200 µl of
water and 2 ml of chloroform/methanol (2:1; v/v). After phase
separation, the radioactivity in the upper phase containing the
phosphocholine was counted in a
-counter.
Acidic and Neutral Ceramidase Assay--
Confluent mesangial
cells were stimulated as described above and homogenized in lysis
buffer containing 50 mM sodium acetate, pH 4.5, 0.5%
Triton X-100, 5 mM MgCl2, 1 mM
EDTA, and 5 mM D-galactonic acid-
-lactone
for the acidic ceramidase, and 50 mM Tris, pH 8.0, 0.5%
Triton X-100, 5 mM MgCl2, 1 mM
EDTA, 5 mM D-galactonic acid-
-lactone for
the neutral ceramidase. Cell homogenates were centrifuged for 10 min at
14,000 × g, and the supernatant was taken for an in vitro assay. 100 µg of protein in a total volume of 100 µl was incubated for 20 h at 37 °C with 20 nCi of
[14C]ceramide. Thereafter, the reaction was stopped by
the addition of 200 µl of water, and lipid extraction was performed
by addition of 2 ml of chloroform/methanol (2:1; v/v). The lower phase
was concentrated and lipids were resolved by TLC using
chloroform/methanol/ammonia (90:20:0.5; v/v) as a solvent. Spots
corresponding to ceramide and sphingosine were analyzed and quantitated
using a Berthold (Nashua, NH) LB 2842 automatic TLC scanner.
 |
RESULTS |
It is well documented from our previous studies that glomerular
mesangial cells and endothelial cells are able to undergo apoptosis
upon stimulation with exogenous NO donors (9, 10). However the exact
mechanism by which the short-lived gas NO mediates its apogenic effect
is still poorly understood.
In this study we evaluated the effect of exogenously delivered NO on
the stress-signaling molecule ceramide in mesangial cells. Fig.
1 shows that long term treatment of
mesangial cells for 24 h with different NO donors like MAHMA-NO,
spermine-NO, SNAP, and GS-NO stimulates a dose-dependent
increase in ceramide levels. Time course studies reveal no acute
increase of ceramide within the first 60 min of NO stimulation (Fig.
2A). However, significant increases of ceramide levels occur after 2-4 h of stimulation and
reach maximal levels (3-4-fold increase) after 24 h of
stimulation with GS-NO (Fig. 2B). In contrast, TNF
causes
a very rapid and transient increase in ceramide formation (Fig.
2A) but fails to induce a late-phase increase of ceramide as
it is seen for NO (Fig. 2B).

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Fig. 1.
Concentration dependence of long term
ceramide formation by different NO donors in mesangial cells.
[14C]Serine-labeled mesangial cells were stimulated for
24 h with the indicated concentrations (in mM) of
MAHMA-NO, spermine-NO, SNAP, and GS-NO. Thereafter lipids were
extracted, separated by TLC as described under "Experimental
Procedures," and the spots corresponding to ceramide were quantitated
on a TLC scanner. Data are expressed as percentage of control values
and are means ± S.D., n varies between 3 and 6.
|
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Fig. 2.
Time course of NO- and
TNF -induced ceramide formation in mesangial
cells. [14C]Serine-labeled mesangial cells were
stimulated with either vehicle ( ), GS-NO (2 mM; ), or
TNF (1 nM; ) for short term (A) or long
term (B) periods as indicated. Thereafter lipids were
extracted, separated by TLC as described under "Experimental
Procedures," and the spots corresponding to ceramide were quantitated
on a TLC scanner. Data are expressed as percentage of control values
and are means ± S.D., n = 3-4.
|
|
The NO-induced ceramide production is independent of cGMP formation as
the membrane-permeant cGMP analog dibutyryl cGMP (10 µM--1 mM) does not cause an increased
ceramide generation (data not shown). To further elucidate the
mechanism of NO-induced ceramide production we treated cells with a
phorbol ester, a direct activator of protein kinase C. Phorbol esters
have been reported to block the proapoptotic activity of NO in several
cell types (25, 26). Fig. 3A
shows that the phorbol ester TPA causes a dose-dependent inhibition of NO-induced ceramide formation. In parallel, NO-induced DNA fragmentation is also blocked by increasing concentrations of TPA
(Fig. 3B), thus suggesting a negative regulatory role for PKC on NO-stimulated ceramide formation and apoptosis. To examine whether ceramide elevations are required for NO to trigger apoptosis we
added exogenous C6-ceramide to the cells exposed to
spermine-NO in the presence of TPA. Indeed, as shown in Fig.
3C exogenous ceramide at least partially bypassed the
suppression of NO-induced apoptosis by TPA.

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Fig. 3.
Effect of PKC on NO-induced ceramide
formation (A) and DNA fragmentation
(B) in mesangial cells. A,
[14C]serine-labeled mesangial cells were stimulated for
24 h with either vehicle or 2 mM spermine-NO in the
presence of the indicated concentrations of the phorbol ester TPA.
Thereafter lipids were extracted and separated by TLC as described
under "Experimental Procedures," and the spots corresponding to
ceramide were quantitated on a TLC scanner. Data are expressed as
percentage of control values and are means ± S.D.,
n = 2. B, confluent mesangial cells were
stimulated for 24 h with either vehicle (co) or 2 mM spermine-NO in the
presence of the indicated concentrations of TPA. Thereafter DNA
fragmentation was measured as described under "Experimental
Procedures." Data are expressed as percentage of control values and
are the means ± S.D., n = 3. C,
confluent mesangial cells were stimulated for 24 h with either
vehicle (co) or 2 mM spermine-NO in the presence
of 100 nM TPA and the indicated concentrations of
C6-ceramide. Thereafter DNA fragmentation was measured as
described under "Experimental Procedures." Data are expressed as
percentage of control values and are means ± S.D.,
n = 3.
|
|
To elucidate the differential effects of NO and TNF
on ceramide
formation, we analyzed the activities of the ceramide-generating and
metabolizing enzymes, i.e. the acidic and neutral
sphingomyelinases and the acidic and neutral ceramidases, respectively.
Treatment of mesangial cells with GS-NO reveals chronic activation of
the acidic sphingomyelinase (Fig.
4A) as well as of the neutral
sphingomyelinase (Fig. 4B), although with distinctly
different time patterns. The acidic sphingomyelinase was activated more
rapidly, reaching maximal activity 2 h after addition of GS-NO and
thereafter slowly declining over the next several hours but still
remaining significantly elevated after 24 h of stimulation (Fig.
4A). By contrast, the neutral sphingomyelinase activity
shows a lag period of 4 h before steadily increasing over the next
20 h of GS-NO stimulation. After a very rapid and transient
(10-20 min) increase in neutral sphingomyelinase activity (data not
shown) TNF
also triggers a delayed activation of the acidic and
neutral sphingomyelinases. A lag period of 4-8 h after TNF
stimulation is required before significant chronic increases of acidic
(Fig. 4A) and neutral (Fig. 4B) sphingomyelinase activities are detected which subsequently persisted for 24 h.

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Fig. 4.
Effect of NO and TNF
on acidic and neutral sphingomyelinase activities in mesangial
cells. Confluent mesangial cells were stimulated for the indicated
time periods with either GS-NO (2 mM; ) or TNF (2 nM; ). Thereafter cell extracts were prepared as
described under "Experimental Procedures" and analyzed for acidic
(A) and neutral (B) sphingomyelinase activities.
Results are expressed as percentage of control values and are
means ± S.D., n = 3.
|
|
Strikingly, we observed that TNF
stimulation causes a chronic
up-regulation of acidic and neutral ceramidase activities in mesangial
cells (Fig. 5, A and
B), whereas GS-NO leads to a time-dependent inhibition of acidic and neutral ceramidase activities (Fig. 5, A and B). This opposite behavior may explain why
GS-NO causes a sustained increase in ceramide levels whereas
TNF
-induced changes in sphingomyelinase and ceramidase activities
counterbalance each other leaving ceramide levels unchanged (Fig.
2B).

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Fig. 5.
Effect of NO and TNF
on acidic and neutral ceramidase activities in mesangial
cells. Confluent mesangial cells were stimulated for the indicated
time periods with either GS-NO (2 mM; ) or TNF (2 nM; ). Thereafter cell extracts were prepared as
described under "Experimental Procedures" and analyzed for acidic
(A) and neutral (B) ceramidase activities.
Results are expressed as percentage of control values and are
means ± S.D., n = 3.
|
|
To test whether the inhibitory effect of NO on acidic and neutral
ceramidase activities is caused by a direct modulation of the enzymes,
increasing concentrations of the rapidly decomposing NO donor MAHMA-NO
(t1/2 = 1-2 min) were incubated in vitro
with the cell extracts containing active acidic and neutral
ceramidases. However, up to concentrations of 1 mM
MAHMA-NO, no significant inhibition of either ceramidase activity is
detected thus excluding a direct inhibitory modulation of the enzymes
by NO (data not shown).
We speculated that the lack of sustained ceramide generation by TNF
might be responsible for the failure of cytokines like TNF
or
interleukin 1
to induce mesangial cell apoptosis, despite inducible
NOS (iNOS) up-regulation and high levels of endogenously produced NO
(10, 27). To evaluate this hypothesis, we used an acidic ceramidase
inhibitor N-oleoylethanolamine (28) to deplete acidic
ceramidase activity in mesangial cells. Inhibition of ceramide
metabolism will lead to increased ceramide levels and given the case
that ceramide is responsible for initiating apoptosis, we expected to
get an increased apoptotic response. Indeed, as shown in Fig.
6, in the presence of
N-oleoylethanolamine, mesangial cells display an enhanced
rate of apoptosis when stimulated with TNF
.
N-Oleoylethanolamine alone had no effect on apoptosis per se.

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Fig. 6.
Effect of
N-oleoylethanolamine on
TNF -induced DNA fragmentation in mesangial
cells. Confluent mesangial cells were stimulated for 24 h
with TNF (2 nM) in the absence or presence of the
indicated concentrations of N-oleoylethanolamine
(NOE). Thereafter DNA fragmentation was measured as
described under "Experimental Procedures." Data are expressed as
percentage of control values and are means ± S.D.,
n = 3. *, p < 0.05, statistically
significant difference compared with TNF -stimulated control.
|
|
 |
DISCUSSION |
The majority of physiological functions of NO appears to be
mediated by activation of its main intracellular receptor guanylate cyclase (29, 30). By nitrosation of the heme moiety NO increases guanylate cyclase activity by 2 orders of magnitude (31, 32). The
generation of cyclic GMP and the subsequent regulation of cyclic
GMP-dependent protein kinases, cyclic GMP-regulated cyclic nucleotide phosphodiesterases, and ion channels is responsible for
functional cell responses triggered by NO (29, 30). However, under
pathological conditions, when large quantities of NO are generated,
other reactions become more prominent and predominate. Among those are
interactions of NO with iron-sulfur centers, protein thiols, lipids,
and zinc fingers in various important cellular molecules (33). Another
important reaction partner of NO is superoxide, and the subsequent
generation of peroxynitrite, which, in the presence of a metal catalyst
(like Fe3+), can be converted to nitronium ion that readily
nitrates tyrosine residues in proteins, and thus may block critical
phosphorylation reactions in signal transduction cascades (34). These
latter reactions may be responsible for the pathophysiological roles played by large amounts of NO produced by the inducible isoform of NOS
that contributes to inflammatory and autoimmune diseases and to host
defense mechanisms required to successfully cope with microbial
infections. The number of newly discovered targets of NO is steadily
increasing, and NO was shown to affect crucial intracellular signaling
pathways. In Jurkat T cells, human umbilical vein endothelial cells,
and in mesangial cells NO was found to activate distinct subgroups of
mitogen-activated protein kinases including the stress-activated
protein kinases and p38 kinase (9, 35).
In this study we demonstrate for the first time that NO triggers
ceramide formation in glomerular mesangial cells in a cyclic GMP-independent manner. This observation provides an unexpected link
between two highly versatile signaling molecules. The sphingomyelin pathway is a ubiquitous signaling pathway that is used by an increasing number of cell surface receptors and environmental stress factors and
includes the generation of ceramide by acidic or neutral
sphingomyelinases (20, 21). Depending on the cell type ceramide was
shown either to trigger cell differentiation or proliferation or to
initiate programmed cell death (apoptosis) (20, 21). Moreover,
stress-induced apoptosis was reported to require ceramide-induced
signaling via the stress-activated protein kinase cascade (36). Our
data now provide evidence for cross-communication between the NO
signaling system and the sphingolipid signaling pathway. Strikingly, we found that NO increases ceramide levels in the cell by a dual mechanism; on the one hand it stimulates the ceramide-producing enzymes
acidic and neutral sphingomyelinases, thus leading to an increased
ceramide formation, and on the other hand NO inhibits the
ceramide-metabolizing enzymes, i.e. acidic and neutral
ceramidases, and thereby prevents degradation of ceramide resulting in
an amplified increase in ceramide steady-state levels. By contrast,
TNF
, besides activating the sphingomyelinases like NO does, causes a
potent activation of the ceramidases, which fully compensates for the increased generation of ceramide, and no net change in chronic ceramide
production is observed. This may also explain why mesangial cells
undergo apoptosis when NO is delivered by exogenous sources but are
resistant to endogenously produced NO after cytokine-induced inducible
NOS expression (10, 27). Activation of both acidic and neutral
ceramidase activities has also been reported to occur in interleukin
1
-exposed rat hepatocytes (37).
It is tempting to speculate that NO either directly or indirectly
affects one of the ceramide-producing or -metabolizing enzymes, i.e. acidic or neutral sphingomyelinases or ceramidases.
Work is presently in progress to elucidate possible direct effects of
NO on enzymes in the ceramide signaling cascade. Whether the NO-induced
formation of ceramide is causally related to apoptosis of mesangial
cells seen after exposure to NO donors (9, 10) remains to be
elucidated. It is however noteworthy that it is not the formation of
ceramide per se but rather the prolonged and
sustained increase of ceramide levels that correlates with programmed
cell death. This becomes obvious after inhibition of acidic ceramidase
by N-oleoylethanolamine (28), which enables mesangial
cells to undergo apoptosis after TNF
stimulation (Fig. 6). However,
it should be noted that this inhibitor has only been characterized in a
few systems, and data on the selectivity of the compound in mesangial
cells are presently not available. Inhibition of ceramidase now unveils
the stimulatory action of TNF
on acidic and neutral
sphingomyelinases and gives rise to sustained increases in ceramide
levels in mesangial cells, which may be required to trigger apoptosis.
In Fig. 7 we have compiled data on
ceramide production in and apoptosis of mesangial cells in response to
a number of different stimuli. There is a strong correlation between
chronic ceramide production and apoptosis of the cells. However,
further work is required to formally establish a causal link between
these two phenomenons.

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Fig. 7.
Correlation between ceramide formation and
induction of apoptosis in mesangial cells. Mesangial cells were
stimulated for 24 h with either DMEM (control), TNF (2 nM), TNF plus NOE (1 mM), MAHMA-NO (2 mM), spermine-NO (2 mM), SNAP (1 mM), and staurosporine (stauro, 100 nM). Thereafter ceramide formation and cell apoptosis were
measured as described under "Experimental Procedures." Data are
means ± S.D. (n = 3-6). A linear regression was
performed and revealed a correlation coefficient between ceramide
formation and apoptosis of 0.88.
|
|
Another interesting aspect of this study is the negative regulatory
role of PKC in NO-induced ceramide formation. Previously, we have shown
that mesangial cells express four PKC isoenzymes, PKC-
, -
, -
,
and -
(38, 39). Moreover, we demonstrated that ceramide is able to
directly interact with PKC-
and -
, but not with PKC-
and -
isoenzymes (40, 41). These data together with the results presented in
this study raise the intriguing possibility of PKC-
and/or PKC-
acting as negative regulators of the sphingomyelin cycle, in a way
similar to the PKC-
-mediated feedback regulation of
hormone-stimulated phosphoinositide turnover (38, 42-44).
Interestingly, C2-ceramide-induced internucleosomal DNA
fragmentation in U937 cells could be blocked by treatment of cells with
phorbol ester (45), thus providing further evidence for a link between
NO-induced ceramide production and subsequent apoptosis. The direct
target of PKC mediating this negative feedback regulation is not yet
known. It will be important though to see whether the NO-induced
ceramide signal is further propagated along the different signaling
pathways thought to be targeted by this lipid and to be crucially
involved in a cell's decision to live or to die.
 |
FOOTNOTES |
*
This work was supported by a fellowship of the Swiss
National Science Foundation (to A. H.), Grant SFB 553 of the Deutsche Forschungsgemeinschaft, and grants from the Cilli-Weil-Stiftung (to A. H.), the Wilhelm-Sander-Stiftung, and the Commission of the European
Union (Biomed 2) (to J. P.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Institute of
Pharmacology and Toxicology, Klinikum der J. W.
Goethe-Universität, Theodor-Stern-Kai 7, D-60590 FrankfurtamMain,
Germany. Tel.: 49-69-63-01-69-63; Fax: 49-69-63-01-79-42; E-mail:
huwiler{at}em.uni-frankfurt.de.
 |
ABBREVIATIONS |
The abbreviations used are:
NO, nitric oxide;
NOS, nitric-oxide synthase;
TNF
, tumor necrosis factor
;
DMEM, Dulbecco's modified Eagle's medium;
MAHMA-NO,
(Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate;
spermine-NO, (Z)-1-{N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]amino}-diazen-1-ium-1,2-diolate;
GS-NO, S-nitrosoglutathione;
SNAP, S-nitroso-N-acetyl-D,L-penicillamine;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
PKC, protein
kinase C.
 |
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