(Received for publication, September 29, 1995)
From the
Recently the sphingomyelin cycle, involving the hydrolysis of membrane sphingomyelin by an activated sphingomyelinase to generate ceramide, has emerged as a key pathway in cell differentiation and apoptosis in leukemic and other cell types. Here we investigate a role for this pathway in the senescence of WI-38 human diploid fibroblasts (HDF). We found that endogenous levels of ceramide increased considerably (4-fold) and specifically (compared with other lipids) as cells entered the senescent phase. Investigation of the mechanism of increased ceramide led to the discovery that neutral sphingomyelinase activity is elevated 8-10 fold in senescent cells. There were no changes in sphingomyelinase activity or ceramide levels as HDF entered quiescence following serum withdrawal or contact inhibition. Thus, the activation of the sphingomyelinase/ceramide pathway in HDF is due to senescence and supports the hypotheses that senescence represents a distinct program of cell development that can be differentiated from quiescence. Additional studies disclosed the ability of ceramide to induce a senescent phenotype. Thus, when exogenous ceramide (15 µM) was administered to young WI-38 HDF, it produced endogenous levels comparable to those observed in senescent cells (as determined by metabolic labeling studies). Ceramide concentrations of 10-15 µM inhibited the growth of young HDF and induced a senescent phenotype by its ability to inhibit DNA synthesis and mitogenesis. These concentrations of ceramide also induced retinoblastoma dephosphorylation and inhibited serum-induced AP-1 activation in young HDF, thus recapitulating basic biochemical and molecular changes of senescence. Sphingomyelinase and ceramide may thus be implicated as mediators of cellular senescence.
Cellular senescence is defined as the limited capacity of cells to undergo population doublings(1) ; consequently, cells have a finite life span beyond which they can no longer proliferate. This finite life span correlates with the age of the organism and with the life expectancy of the species from which the cells were obtained; such that the older the age or the shorter the life span, the less the ability of the cells to undergo population doubling(1) .
Several important observations have been made in understanding the senescent phenotype. Senescence is a dominant process as demonstrated by cell fusion experiments demonstrating that the resultant heterokaryons have a finite life span (2) and by the presence of factors from senescent cells that inhibit DNA synthesis in young cells (3) . Senescence also appears to be an ``irreversible'' process, although it may be overridden by DNA tumor viruses leading to proliferation(4) . Several known biochemical parameters of senescence are beginning to shed light on the underlying mechanisms involved in this developmental program. These include lack of c-fos transcription (5) and AP-1 activation(6) , presence of the Rb protein in a predominantly dephosphorylated form(7) , and the occurrence of several alterations in cell cycle proteins(8, 9, 10) , leading to inhibition of DNA synthesis and lack of cell cycle progression.
Little is known
about signal transduction pathways in cell senescence and less is known
about lipid-mediated signaling pathways in senescence. We have recently
demonstrated that senescent cells have a defect in the phospholipase
D/diacylglycerol/protein kinase C pathway(11) . Ceramide, a key
molecule in sphingolipid metabolism and a candidate second messenger,
has been shown to inhibit phospholipase D (11, 12, 13) . This may implicate the
recently described sphingomyelin cycle in cell senescence. This novel
biochemical pathway has been shown to regulate cell differentiation and
apoptosis(14) . In this cycle, sphingomyelin is hydrolyzed by a
neutral sphingomyelinase, and ceramide is generated in response to a
number of extracellular inducers. Ceramide acts as a second messenger
to mediate many of the effects of these inducers(14) . Here we
investigate the role of the sphingomyelinase/ceramide pathway in
cellular senescence of fibroblasts, the prototypic model of senescence.
We demonstrate that ceramide is a potent inhibitor of growth in young
WI-38 HDF. ()We also demonstrate that endogenous ceramide
levels and a neutral magnesium-dependent sphingomyelinase activity are
markedly elevated in senescent cells. We show that exogenous ceramide
is able to induce a senescent phenotype in young HDF at concentrations
that mimic endogenous levels in senescent cells. This was demonstrated
by the ability of ceramide to inhibit DNA synthesis, AP-1 activation,
and Rb phosphorylation; all important biochemical markers of
senescence. We propose that ceramide is a mediator of cellular
senescence.
We utilized WI-38 HDF at various PD. Young HDF (PD
20-30) were able to grow rapidly in culture as indicated by their
ability to incorporate thymidine in response to serum stimulation into
newly synthesized DNA. Senescent cells (PD 55-60) were unable to
proliferate as indicated by their inability to grow even at 3 weeks
after seeding and by their inability to incorporate significant
[H]thymidine into DNA in response to serum (Fig. 1A).
Figure 1:
Ceramide inhibits growth of young WI-38
HDF and induces cell death. A, young WI-38 HDF, unlike their
senescent counterparts, are able to proliferate as indicated by their
ability to undergo [H]thymidine incorporation
into newly synthesized DNA in response to serum stimulation.
Experiments were performed in triplicate, and the data are
representative of three experiments. B, growth of young WI-38
HDF is progressively inhibited by D-erythro-C
-ceramide (5 (
), 10 (
), 20
(
), or 30 µM (
)). Growth is expressed as the
percentage of control. The data are from single point determinations
and are representative of three experiments. C, top
panel, apoptosis of young HDF is induced by high concentrations of
C
-ceramide. The data are from one experiment performed in
duplicate and are representative of at least two experiments. *, p < 0.05; **, p < 0.001. Bottom panel, cell
death was quantitated by trypan blue staining as described under
``Experimental Procedures.'' The data are from one experiment
performed in triplicate and are representative of two
experiments.
Ceramide potently inhibited growth of
WI-38 HDF. Young, rapidly proliferating cells grown in the presence of D-erythro-C-ceramide (a cell-permeable ceramide),
at concentrations of 5-15 µM, underwent complete
growth arrest analogous to senescent cells (Fig. 1B).
High concentrations of C
-ceramide appeared cytotoxic,
whereby concentrations of greater than 20 µM induced
apoptosis as measured by thymidine release (Fig. 1C, top) and as confirmed by trypan blue uptake (Fig. 1C, bottom).
Because ceramide was able to cause growth arrest in WI-38 HDF and induce terminal differentiation in HL-60 cells (27) and because senescent cells appear to behave similar to growth-arrested and terminally differentiated cells(28) , we investigated the hypothesis that sphingomyelinase and endogenous ceramide may play a role in inducing cell senescence. Initially, we measured ceramide levels in young, old, and senescent WI-38 HDF and correlated ceramide levels to PD elapsed. Ceramide levels remained stable with increasing population doublings as long as the cells were able to proliferate. The capacity to proliferate may also be quantitated as the number of PD remaining. In the experiments shown in Fig. 2, HDF demonstrated a finite PD of 61 after which cells remained metabolically active but failed to grow or take up thymidine. Fig. 2A shows that ceramide levels in young cells (40 PD remaining) were 3.5 ± 0.05 pmol/nmol lipid phosphate. As the PD remaining decreased to 20, ceramide levels remained unchanged. However, when PD remaining decreased further, ceramide levels began to increase to 6.0 ± 0.5 pmol/nmol lipid phosphate. When all cells became senescent, ceramide increased to 14.7 ± 0.8 pmol/nmol lipid phosphate or 4.2-fold over young cells. On the other hand, diacylglycerol levels measured concurrently with ceramide levels increased only modestly with cellular senescence (Fig. 2A). These levels are corrected for total membrane phospholipid content and therefore reflect specific changes in ceramide. In addition, sphingomyelin mass measurements were performed on young and senescent Wi38 HDF and were 58.6 ± 2.9 and 42.5 ± 2.9 pmol/nmol lipid phosphate, respectively.
Figure 2:
Endogenous ceramide levels and neutral
sphingomyelinase activity increase in cell senescence but not in
quiescence. Cells at increasing population doublings were assayed for
levels of endogenous ceramide, diacylglycerol, neutral, and lysosomal
acidic sphingomyelinase activity. A, ceramide levels increase
slowly with increasing population doublings and peak at senescence
(represented by the last point). This was attained when cells showed no
growth for four weeks (i.e. no remaining PD) and did not
incorporate [H]thymidine (see Fig. 1A). In contrast, diacylglycerol levels increased
only modestly with cellular senescence. The experiment shown was
performed in duplicate and is representative of three separate
experiments. B, neutral magnesium-dependent sphingomyelinase
activity is significantly increased in senescent HDF whereas acidic
sphingomyelinase activity appears relatively unchanged. The data
represent the means ± standard error of two duplicate
experiments. C, ceramide levels do not change significantly
between exponentially growing (EG), quiescent serum-deprived (SD), or contact-inhibited (CI) HDF. The data for EG and SD represent the mean ± S.E. from three
separate experiments performed in duplicate. CI represents the
mean ± range from one experiment performed in duplicate. D, neutral sphingomyelinase activity remains unchanged in EG, SD, or CI HDF. Blank, no
enzyme. The data represent the mean ± range from one experiment
performed in duplicate.
A neutral
sphingomyelinase has been implicated in the generation of ceramide in
response to inducers of differentiation and growth arrest (29, 30, 31) . Indeed, we demonstrate an
8-fold increase in neutral magnesium-dependent sphingomyelinase
activity in protein extracts from senescent cells (Fig. 2B) as compared with young HDF. In contrast, acid
sphingomyelinase, a well characterized lysosomal enzyme(32) ,
had higher base-line activity in young HDF but increased only modestly
in senescent extracts (Fig. 2B). These remarkable
increases in ceramide levels, in ceramide:diacylglycerol ratio, and in
neutral sphingomyelinase activity were even more marked than those
observed in cell differentiation(27) . They also represent
permanent (stable and prolonged) increases in ceramide and
sphingomyelinase activity as opposed to the transient signaling
increases in response to inducers of apoptosis or differentiation such
as tumor necrosis factor (33) and dihydroxyvitamin
D
(27) .
In order to evaluate if those increases
in ceramide levels and sphingomyelinase activity were specific to
senescence or were a consequence of growth arrest, we performed the
same measurements in exponentially growing and in quiescent HDF.
Exponentially growing cells were harvested 2 days after seeding,
whereas quiescent cells were rendered so either by 48 h of incubation
in medium containing 0.1% FBS or by contact inhibition in the presence
of 10% FCS and then harvested. The conditions for quiescence were
determined as follows: WI-38 cells were seeded at 1 10
cells in 10-cm plates. 2 days later the cells were placed in
medium containing 0.1% FBS for 48 h. The cells were then harvested for
determination of either their labeling index or for flow cytometry
studies as described (34) using a Becton Dickinson
FACStar
flow cytometer (San Jose, CA). The cells showed
a labeling index of <2%, and flow studies demonstrated that 97% of
the cells were in the G
phase of the cell cycle. Cells
allowed to become quiescent by contact inhibition in the presence of
10% FCS demonstrated a similar cell cycle profile. Upon determination
of ceramide levels (Fig. 2C), we demonstrated that
there was no difference between quiescent and exponentially growing
young HDF; also there was no difference in neutral sphingomyelinase
activity between quiescent and exponentially growing young HDF (Fig. 2D). Therefore, the changes in sphingomyelinase
and ceramide seen were specific to senescence and not a mere
consequence of growth arrest.
These increases in ceramide levels and
sphingomyelinase activity coupled with the inability of senescent cells
to respond to mitogenic stimuli led us to investigate whether ceramide
imparts the senescent phenotype to young HDF. We examined several
biological and biochemical parameters of senescence. Senescent cells,
unlike young quiescent cells, are unable to undergo DNA synthesis and
are unable to divide in response to serum or growth factor stimulation (Fig. 1)(28) . In order to mimic the persistent
elevation of endogenous ceramide in senescent cells, young HDF were
treated with concentrations of exogenous C-ceramide for
1-3 days, which resulted in a persistent elevation in cellular
ceramide levels comparable to those seen in senescent cells. This was
demonstrated by uptake and metabolism studies utilizing
[
C]hexanoyl-sphingosine (15 µM) (Table 1) or hexanoyl-[
H]sphingosine (not
shown). The cells took up 16% of the label within 1 h. By 24 and 48 h,
44 and 63%, respectively, of the C
-ceramide radiolabel (Rf
0.73 in chloroform:methanol (80:20)) was metabolized to two predominant
compounds, Rf = 0.37 and 0, which were observed first as
cell-associated but then accumulated in the medium. These products have
not been identified but do not appear to be the simple sphingosine,
triglyceride, sphingomyelin, or glycosphingolipid metabolites.
Membrane-associated radiolabel was 35% ceramide at 24 h and only 26% by
48 h. To compare effective added C
-ceramide levels, we
calculated the ratio of C
-ceramide to total lipid. At 24 h,
cell membranes contained 22 pmol of ceramide/nmol of lipid phosphate.
This decreased to 18 pmol/nmol of lipid phosphate by 48 h. These levels
closely resemble endogenous ceramide levels found in senescent
fibroblasts (see Fig. 2A).
We next elected to
evaluate the ability of ceramide to impart senescence by evaluating
inhibition of DNA synthesis, a known biological parameter of
senescence. C-ceramide (5-20 µM)
treatment for 24 h progressively inhibited
[
H]thymidine incorporation in young HDF in
response to serum (Fig. 3A). There was no difference in
the degree of inhibition of new DNA synthesis if the cells were kept in
the presence of ceramide for up to 60 h (data not shown). These data
demonstrate that ceramide is a potent inhibitor of DNA synthesis in
young HDF. When C
-ceramide was washed out with 10%
FCS-containing medium, intracellular ceramide levels returned to base
line as documented by studies using
C-labeled
C
-ceramide, and the cells were able to resume growth and
undergo DNA synthesis at the same rate as control cells (data not
shown). This raised the intriguing possibility that senescence may be
reversible if ceramide levels can be brought down to levels seen in
young cells.
Figure 3:
Ceramide inhibits DNA synthesis. Young
WI-38 HDF were treated for 24 h with C-ceramide (5-20
µM) in the absence or presence of PDMP (20 or 50
µM) (A) or 5 or 10 µM of
C
-ceramide, dihydro-C
-ceramide, or
dioctanoylglycerol (B). Thymidine incorporation in response to
24 h of serum stimulation was determined as described under
``Experimental Procedures.'' The data are from one experiment
performed in triplicate and are representative of two separate
experiments.
Because a family of ceramide metabolites,
glycosphingolipids, have been reported to be growth
inhibitory(35) , we used a ceramide glycosylation inhibitor,
PDMP(36) , to rule out the possibility that
C-ceramide was acting through one of these products.
Although PDMP alone had little effect up to 20 µM, it was
growth inhibitory at 50 µM (Fig. 3A). PDMP
at 20 µM had little effect on
C
-ceramide-mediated DNA synthesis inhibition, but at 50
µM the PDMP effect was additive with
C
-ceramide (Fig. 3A). This indicates that
glycosylation of C
-ceramide is not required for growth
inhibition.
To test the specificity of this effect to
C-ceramide, cells were treated in parallel with
C
-ceramide, 4,5-dihydro-C
-ceramide, and
dioctanoylglycerol. Fig. 3B demonstrates that DNA
synthesis is not inhibited by dihydro-C
-ceramide, which is
desaturated at the 4,5 double bond and that the glycerol based analog
is also inactive in this assay.
In order to determine if the
inhibition of DNA synthesis induced by ceramide imparted a senescent
phenotype, we measured labeling indices in young HDF in the presence of
ceramide. Young HDF were able to label 67.5 ± 9.5% of their
nuclei by [H]thymidine in response to serum
stimulation (Fig. 4A). In contrast, senescent cells had
a labeling index of <2% (Fig. 4B), cells grown in 10
µM C
-ceramide had a labeling index of 12
± 5% (Fig. 4C), and cells grown in 15 µM C
-ceramide had a labeling index of 2.5 ± 0.5% (Fig. 4D) and morphologically resembled senescent
cells.
Figure 4:
Ceramide induces a senescent phenotype as
determined by labeling indices. A, young WI-38 HDF were serum
deprived for 48 h followed by stimulation with 10% FBS for 48 h. This
resulted in a labeling index (% cells with stained nuclei) of 75%. (B)
Senescent WI-38 HDF did not grow for at least four weeks in culture
after seeding. They were stimulated with 10% FBS for 48 h and had a
labeling index of <2%. C and D, young cells were
treated with C-ceramide (10 µM) (C)
or 15 µM (D) for 48-72 h and then
serum-stimulated as above, resembled senescent cells morphologically.
Labeled nuclei were counted and expressed as the percentage of total
nuclei. 500 nuclei were counted from random fields. The data are from
single determinations and are representative of three
experiments.
Several biochemical parameters of cell aging and cellular
senescence have been defined(28) . An important discovery came
with the finding that senescent cells cannot undergo AP-1 activation in
response to mitogenic stimuli(6) , presumably because of an
inability to transcribe c-Fos(5) . This may be an important
mechanism in preventing senescent cells from entering the cell cycle.
Young HDF treated with C-ceramide (10 µM) were
unable to undergo AP-1 activation in response to serum stimulation as
demonstrated by gel shift analysis of specific AP-1 complexes. Fig. 5A demonstrates that upon serum stimulation young
HDF are able to respond by activation of AP-1 (lanes 1 and 2), whereas ceramide-treated cells (lanes 5 and 6) like senescent cells (lanes 7 and 8) were
unable to respond by activation of AP-1. The specificity of AP-1
binding was demonstrated by the ability of cold wild type probe to
compete off the complex (Fig. 5A, lane 3) as
well as the lack of complex formation with a mutated AP-1 probe (Fig. 5A, lane 4) (see ``Experimental
Procedures'' for exact sequences). Fig. 5B demonstrates the presence of Fos protein in the complex found in
young but not senescent or ceramide-treated cells. In other studies
from our laboratory we showed that ceramide inhibits phospholipase D
activation leading to lack of diacylglycerol generation and protein
kinase C activation in young HDF in response to serum stimulation,
again mimicking alterations occurring in cellular
senescence(11) . These studies demonstrate that ceramide
accumulation inhibits mitogenic stimulation, similar to what occurs in
senescent cells.
Figure 5:
Ceramide inhibits AP-1 activation. A, gel shift analysis utilizing a labeled AP-1 oligonucleotide
and nuclear extracts obtained from young quiescent WI-38 HDF (lane
1), young serum-stimulated (YSS) WI-38 HDF demonstrating induction
of AP-1 binding (lane 2), YSS extract preincubated with cold
wild type AP-1 oligonucleotide demonstrating specificity of binding (lane 3), YSS extract incubated with labeled mutant AP-1
oligonucleotide demonstrating no specific binding (lane 4),
young quiescent extract from cells pretreated with
C-ceramide (10 µM) (lane 5), YSS
extract from cells pretreated with C
-ceramide (10
µM) demonstrating no induction of AP-1 binding (lane
6), senescent WI-38 HDF (lane 7), and senescent
serum-stimulated WI-38 HDF demonstrating no induction of AP-1 binding (lane 8). B, the AP-1 complex contains c-Fos: YSS
extract (lane 1) or YSS extract preincubated with c-Fos
antibody at 4 °C for 30 min (lane 2) partial competion
AP-1 binding.
Another important biochemical marker came with the
discovery that senescent HDF are unable to phosphorylate the
retinoblastoma protein in response to mitogenic stimuli(7) .
Remarkably, C-ceramide in concentrations ranging from 3 to
20 µM was able to progressively inhibit Rb phosphorylation
in young HDF in response to serum stimulation as indicated by Western
blot analysis (Fig. 6, top). In addition,
C
-ceramide (15 µM) was able to induce complete
Rb dephosphorylation by 20-24 h, thus mimicking senescent HDF (Fig. 6, bottom).
Figure 6:
Ceramide inhibits Rb phosphorylation and
induces Rb dephosphorylation. Quiescent young WI-38 HDF treated with
increasing concentrations of ceramide were progressively unable to
undergo Rb phosphorylation in response to serum stimulation.
Exponentially growing cells (EG), serum-deprived cells (SD), and serum-deprived and restimulated cells treated with
ethanol vehicle or the indicated concentration of
C-ceramide were harvested. The proteins were analyzed by
gel electrophoresis and immunoblotted with anti-Rb antisera. EG cells showed various forms of Rb phosphoprotein. Rb in SD cells became completely dephosphorylated. Upon serum stimulation,
cells were able to phosphorylate Rb. However, cells treated with
increasing concentrations of ceramide were unable to respond to serum
stimulation, and Rb remained dephosphorylated (top panel).
C
-ceramide (15 µM) induced complete Rb
dephosphorylation by 20-24 h in young WI-38 HDF, thus mimicking
senescent cells (SC) (lower
panel).
In this study, evidence is provided for a role for ceramide in cellular senescence. First, ceramide levels and sphingomyelinase activity are significantly and specifically elevated in senescent cells and not in quiescent cells. Indeed, the changes in sphingomyelinase and ceramide appear to specifically distinguish senescence from quiescent growth arrest in WI-38 HDF. Second, the addition of ceramide to young HDF recapitulates many of the established parameters of cell senescence. These include the ability of ceramide to: 1) inhibit DNA synthesis; 2) inhibit growth; 3) inhibit AP-1 activation; and 4) activate Rb through dephosphorylation. Taken together the activation of sphingomyelinase and the ability of ceramide to induce parameters of senescence begin to point to a role for this pathway in the regulation of senescence.
These remarkable increases in ceramide levels, in the
ceramide:diacylglycerol ratio, and in neutral sphingomyelinase activity
are even more marked than those observed in cell
differentiation(27) . They also represent permanent (stable and
prolonged) increases in ceramide and sphingomyelinase activity as
opposed to the transient signaling increases in response to inducers of
apoptosis or differentiation, such as tumor necrosis factor (33) and dihydroxyvitamin D
(27) .
Remarkably, there is no difference in ceramide levels or in
sphingomyelinase activity between quiescent and exponentially growing
young HDF. Therefore, the changes in sphingomyelinase and ceramide seen
in the WI-38 cells are specific to senescence and not a mere
consequence of growth arrest.
The elevation of sphingomyelinase
appeared to be specific to the neutral, Mg-dependent
enzyme. This enzyme has been shown to be specifically (compared with
the acidic enzyme) activated by various agonists(14) .
Sphingomyelin and particularly the hydrolyzable pool of sphingomyelin
has been shown to be concentrated in the plasma membrane(37) ,
where it would be accessible to the neutral,
Mg
-dependent sphingomyelinase. Although the acid
sphingomyelinase exhibits a higher specific activity in the WI-38
cell-free assay, it does not appear to be significantly increased in
senescent cells. This enzyme is sequestered in the lysosomes that
generally function in a degradative or recycling capacity. It is known
that Nieman-Pick cells, which lack the acid sphingomyelinase,
accumulate sphingomyelin in their lysosomal membranes (38) .
These cells also undergo senescence, (
)indicating that acid
sphingomyelinase is not necessary for WI-38 senescence. Entry of cells
into senescence is accompanied by induction of neutral
sphingomyelinase, although we do not know the mechanism of that
activation. However, many changes occur during cellular senescence
(reviewed in (39) ), such as decreased membrane fluidity,
increased protein oxidation, decreased DNA methylation, telomere
shortening, and defects in mitogenic signaling; one or more of these
events may act as a signal for activation of sphingomyelinase.
The
ability of exogenously administered ceramide to induce a senescent
phenotype raises the possibility that one of its metabolites could be
the biologically active species. We observe the formation of two minor
metabolites, which are produced over the course of several hours.
Hexanoyl-[H]sphingosine (data not shown) and
[
C]hexanoyl-sphingosine both showed the
production of these same two spots on TLC. Our preliminary analysis
demonstrates that these compounds do not represent predictable products
such as glycosphingolipids, sphingosine, or sphingomyelin. In this
context Rani et al.(40) have demonstrated that PDMP
induces cell cycle arrest in NIH-3T3 fibroblasts; however, in that
study this effect was not totally mimicked by exogenous ceramide,
probably due to administration of C
-ceramide in
serum-containing medium, which attenuates its effects(41) .
However, because ceramide is elevated selectively in senescence, we
feel that ceramide is either the active species itself or an immediate
precursor of the active species.
Several molecular changes that
occur with cellular senescence have been characterized(28) . In
this study we demonstrate that several of these biochemical events
occur in response to C-ceramide. The inability of senescent
cells to activate the transcription factor AP-1 in response to
mitogenic stimuli (6) appears to be one of the critical events
in preventing senescent cells from entering the cell cycle. Young HDF
treated with C
-ceramide were unable to undergo AP-1
activation in response to serum stimulation. In a previous study we
found that a mitogenic pathway mediated by PLD activation and DAG
generation is inhibited in senescent HDF and that
C
-ceramide inhibits this pathway(11) , again
mimicking alterations occurring in cellular senescence. Another
important biochemical marker is that senescent HDF are unable to
phosphorylate the retinoblastoma protein in response to mitogenic
stimuli(7) . This may be due to the overexpression of the
recently described senescent cell-derived inhibitor of DNA synthesis
(Sdi1 or p21), a protein involved in binding and inhibiting the kinase
activity of cyclin-Cdk complexes(10) . Remarkably,
C
-ceramide was able to inhibit Rb phosphorylation in young
HDF in response to serum stimulation and to induce complete Rb
dephosphorylation; in addition preliminary data from our laboratory
demonstrate that ceramide is also able to induce Sdi1 protein levels.
Thus the addition of ceramide has a dual role in inducing a senescent
phenotype in HDF by: 1) activation of a growth suppressor pathway
through Rb dephosphorylation and 2) inhibition of the mitogenic pathway
mediated by c-Fos and AP-1. Although sphingomyelinase activation and
ceramide generation may not ``cause'' senescence, our data
presented here support a role for ceramide in mediating or maintaining
senescent cells in a nonreplicative state.
Ceramide has been shown to activate a protein phosphatase 2A-like protein phosphatase termed ceramide-activated protein phosphatase(42) . Okadaic acid has been shown to inhibit ceramide-activated protein phosphatase, to be mitogenic, and to partially reverse cellular senescence(24) . This allows us to speculate that inhibition of the sphingomyelinase/ceramide pathway might reverse cell senescence. Our studies showing that washing out ceramide allows cells to re-enter the cell cycle and escape senescence support this hypothesis. Thus, further understanding of sphingomyelinase and of the mechanism of action of ceramide should allow important insight into the molecular mechanisms involved in cellular senescence.
In summary, cellular senescence is a state of growth arrest that partially resembles quiescent growth arrest. Very few biochemical events are able to distinguish senescence from quiescent growth arrest. Ceramide appears to be one of the first biologically active molecules that do so. We have previously shown that ceramide increases significantly after serum deprivation of Molt-4 cells, which induces a terminal event of growth arrest characterized by apoptosis and cell cycle arrest(43) . The events occurring in senescence are analogous in the sense that senescence is a terminal event. Fibroblast quiescence, on the other hand, is a reversible event, and consequently it is expected that ceramide levels are not elevated. Taken together, significant new evidence now points to ceramide as an inducer of terminal events of cell biology, i.e.. apoptosis, differentiation, and now cellular senescence. These results provide an important first step into a new avenue of research on the biology of senescence and aging.