(Received for publication, July 7, 1995; and in revised form, September 1, 1995)
From the
Ceramide produced by the hydrolysis of sphingomyelin is an
important cellular intermediate in hormone action. Here, we present
evidence that interleukin 1 (IL-1
) binding to normal human
fibroblasts initiates a lipid messenger cascade that takes place in a
sphingomyelin-rich plasma membrane domain with the characteristics of
caveolae. Hormone binding first stimulated the appearance of
diacylglycerol (DAG) in a caveolea-rich membrane fraction isolated from
whole cells. This was immediately followed by the loss of a resident
population of sphingomyelin from the fraction and the concomitant
appearance of ceramide. The ceramide produced in response to IL-1
blocked platelet-derived growth factor-stimulated DNA synthesis.
IL-1
stimulated the appearance of DAG in other fractions from the
same cell, but this DAG was not coupled to ceramide production. This
indicates that ceramide production is highly compartmentalized at the
cell surface. Since caveolae are known to be involved in membrane
internalization, they may be essential for the delivery of ceramide to
a site of action within the cell.
The caveola is a membrane domain that can undergo an internalization cycle. The cycle begins with membrane invagination, which leads to the formation of a plasmalemmal vesicle. These vesicles may migrate toward the center of the cell (1) or remain nearby the cell surface(2, 3, 4) . Plasmalemmal vesicles do not appear to merge with other endocytic pathways as they deliver internalized molecules to either the cytoplasm (5, 6, 7) or to the endoplasmic reticulum (8) of the cell. Eventually the vesicle returns to the cell surface to complete the cycle.
The caveolae internalization cycle
depends on several resident molecules. Cholesterol appears to be a
structural molecule that is necessary for the integrity of the caveolae
membrane coat (9) and the shape of the membrane(10) .
PKC is a resident protein that seems to control membrane
invagination(11, 12) . Caveolae contain a 90-kDa
protein that is a substrate for PKC
. Conditions that remove
PKC
from caveolae prevent phosphorylation of this protein and
block membrane invagination(12) . Finally, caveolae contain a
protein phosphatase that dephosphorylates the 90-kDa
protein(12) . The phosphatase may be a target for drugs that
inhibit the return of vesicles to the membrane(1) .
The
molecular composition of caveolae is different from other membrane
domains. The core membrane structure appears to be enriched in
cholesterol(8, 13, 14) ,
gangliosides(1, 15, 16) , GPI-anchored
membrane proteins(2, 17, 18, 19) ,
and the integral membrane protein caveolin(9, 18) . It
may also be enriched in sphingomyelin(20) . Recent biochemical
and immunoelectron microscopic evidence indicates that caveolae contain
high concentrations of several different signaling molecules. These
include 1) GPI-anchored hormone receptors(21) , 2) inositol
1,4,5-trisphosphate receptor(22) , 3) protein kinase C
(PKC)()(12, 23) , 4) G protein-coupled
membrane receptors(3, 24, 25) , 5) multiple
heterotrimeric GTP binding proteins(17, 18) , 6)
non-receptor tyrosine kinases(18, 26) , 7) an
ATP-dependent Ca
pump(27) , and the epidermal
growth factor receptor(19) . The presence of these molecules
has prompted a search for signaling events that originate in this
location.
There are many ways that the caveolae internalization
cycle could be harnessed for signal transduction(28) . One
possibility is that caveolae are used to compartmentalize the synthesis
of key intermediates in a signaling cascade. Wiegmann et al.(29) have shown that the tumor necrosis factor receptor has two
different domains that can stimulate ceramide production. One domain
acts on a neutral sphingomyelinase and the other on an acid
sphingomyelinase. To do this, tumor necrosis factor must stimulate
ceramide production within different compartments of the same cell. The
high concentration of sphingomyelin that appears to be in caveolae (20) suggests that they could be one of the compartments. To
investigate this possibility, we developed a human fibroblast model
system for studying IL-1-dependent ceramide
production(30) . We now present evidence that IL-1
stimulates the conversion of sphingomyelin to ceramide in a membrane
fraction that has the biochemical characteristics of caveolae. The
ceramide produced in this membrane inhibits DNA synthesis.
Figure 1:
IL-1 inhibits PDGF-stimulated
[
H]thymidine incorporation (A) while
stimulating ceramide formation (B). A, cells were
grown to confluence in 12-well plates, washed once with 1 ml/well PBS,
and incubated in MEM plus 100 µg/ml BSA for 30 h at 37 °C. The
medium was changed, 5 ng/ml PDGF plus the indicated concentration of
IL-1
(
) was added to the dish, and the cells were further
incubated for 12 h. At the end of the incubation, 1 µCi of
[
H]thymidine (0.4 Ci/mmol) was added to each
well, and the cells were incubated further for 6 h at 37 °C.
[
H]Thymidine incorporation was measured as
described. B, cells were grown to confluence in 6-well plates.
For the final 48 h of culture, [
H]palmitate (10
µCi/well) was in the medium. Fresh MEM containing 100 µg/ml BSA
was added to each well, and the cells were incubated for 2 h at 37
°C before the indicated amount of IL-1
was added. At the end
of 1 h, the lipids were extracted from the cells and separated by TLC
as described. The ceramide spot was scraped and counted. Each point is
the mean ± S.D. (n =
3).
IL-1 also caused a dose-dependent increase in the concentration
of ceramide (
, Fig. 1B). Cells were incubated
in the presence of [
H]palmitate to label the
sphingomyelin pool and then exposed to various concentrations of
IL-1
for 1 h. A basal level of [
H]ceramide
occurred in the absence of IL-1
, but this increased up to 2-fold
as the concentration of IL-1
was increased. The response was
maximal at
15 ng/ml IL-1
.
Exogenously added ceramide
suppressed PDGF-stimulated DNA synthesis (Fig. 2). Confluent
fibroblasts cultured for 24 h in the absence of serum incorporated 7
pmol of [H]thymidine per mg of protein (control, Fig. 2A). Incubation of these cells in the presence of
PDGF for 12 h stimulated [
H]thymidine
incorporation nearly 3-fold (PDGF, Fig. 2A). This
increase was completely blocked by the addition of 4 µg/ml
C
-ceramide to the medium (PDGF+ceramide, Fig. 2A). DNA synthesis was suppressed equally well by
20 µg/ml DAG C8:0 (PDGF+DAG, Fig. 2A), a
membrane-permeable diacylglycerol(37) . DAG may have this
effect because in some cells it stimulates ceramide
synthesis(29, 38) .
Figure 2:
IL-1 acts through DAG and ceramide to
suppress [
H]thymidine incorporation in human
fibroblasts. A, cells were cultured in 12-well plates to
confluence, washed once with 1 ml/well PBS, and incubated in MEM
containing 100 µg/ml BSA for 30 h at 37 °C. The medium was
replaced with fresh medium that contained no addition (control), 5
ng/ml PDGF (PDGF), 5 ng/ml PDGF plus 4 µg/ml C
-ceramide
(PDGF+ceramide), or 5 ng/ml PDGF plus 20 µg/ml DAG C8:0
(PDGF+DAG) and then incubated an additional 12 h at 37 °C. At
the end of the incubation, DNA synthesis was measured as described in Fig. 1. B, the medium was replaced with fresh medium
that contained no addition (control), 5 ng/ml PDGF (PDGF), 5 ng/ml PDGF
plus 10 ng/ml IL-1
(PDGF+IL-1
), 5 ng/ml PDGF plus
IL-1
and 10 µg/ml D609 (PDGF+IL-1
+D609), or
PDGF plus D609 and 4 µg/ml C
-ceramide
(PDGF+D609+ceramide). All cells were then incubated for 12 h
at 37 °C before [
H]thymidine (0.4 Ci/mmol)
incorporation was measured. Each point is the mean ± S.D. (n = 3).
To determine if DAG was an
intermediate in IL-1-stimulated ceramide production, we blocked
DAG production with an inhibitor of phosphatidylcholine-specific
phospholipase C(39) . Fig. 2B shows that PDGF
stimulated DNA synthesis 2-fold (PDGF, Fig. 2B), but
the presence of IL-1
in the media completely blocked this increase
(PDGF+IL-
, Fig. 2B). The combination of the
phosphatidylcholine-specific phospholipase C inhibitor D609 (20
µg/ml) and IL-1
in the media prevented IL-1
from
suppressing DNA synthesis (compare PDGF with
PDGF+D609+IL-1
, Fig. 2B). D609 did not
stop C
-ceramide from inhibiting DNA synthesis, indicating
that the drug did not block the signaling activity of ceramide
(PDGF+D609+ ceramide, Fig. 2B). The results
in Fig. 2suggest that DAG is an obligate intermediate in
IL-1
-mediated ceramide formation, but ceramide is the active
signaling molecule.
Figure 3:
Preparation of caveolae-rich fractions.
Cells were grown to confluence in 150-mm dishes. In one set of cells,
[H]arachidonate (10 µCi/dish) was present in
the medium for the last 24 h. The cells were solubilized in 1% Triton
X-100 at 4 °C and fractionated on sucrose gradients as described.
Each fraction was then assayed for protein (A),
[
H]arachidonate-labeled lipids (B), and
both alkaline phosphatase activity and caveolin (C). A, the amount of protein in each fraction was measured as
described. B, the total amount of labeled lipids in each
fraction was measured as described. C, fractions were
collected and either assayed for alkaline phosphatase activity
(alkaline phosphatase) or immunoblotted with anti-caveolin IgG
(caveolin) as described.
One
advantage of this method is that the fractionation procedure allows a
comparison between the caveolae fractions (fractions
4-7) and the remainder of the cell (fractions
11-15). To determine the distribution of sphingomyelin on
this gradient, we cultured cells in the presence of
[H]choline for 48 h before fractionating the
cells and measured the amount of
[
H]choline-labeled sphingomyelin (
, Fig. 4). The majority of the sphingomyelin was in the caveolae
fractions (fractions 4-7). On average, this fraction
contained 50-70% of the total cellular sphingomyelin. Nearly all
of this sphingomyelin was located in the extracellular leaflet of the
plasma membrane because treatment of cells with 0.1 unit/ml of neutral
sphingomyelinase prior to the fractionation procedure removed the
majority of the choline head groups from the sphingomyelin (
, Fig. 4). This suggests that the Triton X-100 insoluble
sphingomyelin did not come from internal membranes of the cells.
Figure 4:
Sphingomyelin is highly enriched in
caveolae fractions. Human fibroblasts were grown to confluence in
150-mm dishes. For the final 48 h of culture,
[H]choline chloride (10 µCi/dish) was present
in the medium. One set of cells was fractionated directly (
),
while the other set (
) was washed with 10 ml per dish of PBS and
incubated in MEM containing 100 µg/ml BSA and 0.1 µ/ml neutral
sphingomyelinase for 1 h at 37 °C. Cells were fractionated as
described above. The total lipids from each fraction were extracted and
separated by TLC as described. The sphingomyelin spot was scraped and
counted.
The
caveolae fraction was also highly enriched in ceramide (Fig. 5).
We labeled cells with [H]palmitate, prepared
sucrose gradient fractions, and measured the ceramide content.
Approximately 50% of all the [
H]ceramide in the
cell was in the caveolae fractions (
, Fig. 5). The
remainder was in the soluble fractions at the bottom of the gradient. A
replicate set of cells that had been incubated in the presence of
IL-1
for 1 h had
50% more [
H]ceramide
in the caveolae fractions than control cells (
, Fig. 5).
By contrast, [
H]ceramide was not increased in the
bottom fractions (compare fractions 11-14, Fig. 5). All of the IL-1
-stimulated increase in ceramide
detected in whole cells could be accounted for by the amount that
appeared in the caveolae fraction (compare
, Fig. 1B, with
, Fig. 5).
Figure 5:
IL-1-dependent appearance of ceramide
in caveolae fractions. Cells were grown to confluence in 150-mm dishes.
During the final 48 h in culture, [
H]palmitate
(60 µCi/dish) was present in the dish. Cells were washed once with
10 ml of PBS per dish and cultured in MEM containing 100 µg/ml BSA
for 2 h at 37 °C. The medium was replaced with fresh medium of the
same composition, and the cells were incubated for 1 h in the presence
(
) or absence (
) of IL-1
(10 ng/ml). Cells were
fractionated, and the total lipids in each fraction were extracted and
separated by TLC. The ceramide spot was scraped and
counted.
The
sphingomyelin in the caveolae fraction was the source of the
IL-1-stimulated increase in ceramide (Fig. 6). Fibroblasts
were cultured in the presence of [
H]palmitate to
label the sphingomyelin. The media were replaced with fresh media
containing 10 ng/ml IL-1
, and the cells were incubated for various
times. The caveolae fractions were prepared at the end of each
incubation, and the amount of both
[
H]sphingomyelin (
, left
ordinate, Fig. 6) and [
H]ceramide
(
, right ordinate, Fig. 6) was measured. With
time in the presence of IL-1
, there was a reciprocal decline in
the amount of [
H]sphingomyelin and an increase in
the amount of [
H]ceramide in this fraction.
Nearly all of the radioactivity appearing in the ceramide lipid at each
time point could be accounted for by the amount of
[
H]sphingomyelin that was lost.
Figure 6:
IL-1 stimulates the loss of
sphingomyelin and an increase in ceramide in caveolae fractions. Cells
were grown to confluence in 150-mm dishes. During the final 48 h in
culture, [
H]palmitate (60 µCi/dish) was
present in the dish. Cells were washed once with 10 ml of PBS per dish
and cultured in MEM containing 100 µg/ml BSA for 2 h at 37 °C.
The medium was replaced with fresh medium of the same composition, and
the cells were incubated for the indicated time in the presence of 10
ng/ml IL-1
. The caveolae fraction (fractions 5-8 from the
sucrose gradient) was collected from sucrose gradients and pooled for
lipid extraction. Lipids were separated by TCL, and the sphingomyelin
and ceramide spots were scraped for counting. Each point is the mean
± S.D. (n = 3).
These results
suggested that the caveolae fraction contains sphingomyelinase
activity. We prepared sucrose gradient fractions and assayed each
fraction for Zn-independent, acid sphingomyelinase
activity (Fig. 7). Most of the activity was in the whole cell
fractions at the bottom of the gradient (fractions
11-15). However, there was a peak of activity in the
caveolae fractions (fractions 4-8). Despite the low
total activity, the specific activity in the peak (fraction
6), caveolae fraction was comparable (25 nmol/mg protein/h) to the
activity in the peak (fraction 14), whole cell fraction (42
nmol/mg protein/h). Very little neutral sphingomyelinase activity was
detected in the caveolae fraction (data not shown).
Figure 7:
Distribution of
Zn-independent acid sphingomyelinase activity in
sucrose density gradient fractions. Cells were grown to confluence in
150-mm dishes and fractionated on sucrose gradients. Each fraction (100
µl) was mixed with 100 µl of
[
C]choline-labeled sphingomyelin prepared as
substrate micelles and incubated for 15 min at 37 °C. The released
[
C]choline was extracted and counted as
described.
Figure 8:
IL-1 stimulates DAG production in
both caveolae and whole cell fractions. Cells were grown to confluence
in 150-mm dishes. During the final 48 h in culture,
[
H]palmitate (60 µCi/dish) was present in the
dish. Cells were washed once with 10 ml of PBS per dish and cultured in
MEM containing 100 µg/ml BSA for 2 h at 37 °C. Cells were not
treated (
), incubated in the presence of IL-1
(10 ng/ml)
for 5 min (
), or incubated for 5 min in the presence of IL-1
plus 50 µg/ml D609 (
). The later set of cells was pretreated
with D609 for 10 min before the addition of IL-1
. The cells were
washed with ice-cold PBS before fraction on sucrose gradients. Lipids
from each fraction were extracted and separated by TLC. The DAG spot
was scraped and counted.
If the DAG that appeared in the
caveolae fraction was coupled to ceramide synthesis(29) , then
D609 should prevent the IL-1-dependent appearance of ceramide in
this fraction (Fig. 9). When
[
H]palmitate-labeled cells were incubated in the
presence of IL-1
for 5 min, the level of
[
H]ceramide increased in the caveolae fractions
but not other fractions in the gradient (compare
with
, Fig. 9A). The increase was more modest than seen in
other experiments owing to the shorter incubation time. The
IL-1
-dependent appearance of [
H]ceramide was
completely blocked by D609 (compare
with
, Fig. 9B). We also incubated a replicate set of cells in
the presence of DAG C8:0 for 5 min (Fig. 9C) to see if
exogenous DAG could mimic the effects of IL-1
. Surprisingly, the
[
H]ceramide level was only elevated in the
caveolae fractions (compare
with
, Fig. 9C). Therefore, DAG specifically stimulates the
conversion of sphingomyelin to ceramide in the caveolae fraction of the
plasma membrane.
Figure 9:
IL-1 and DAG only stimulate ceramide
production in caveolae fractions. Cells were grown to confluence in
150-mm dishes. During the final 48 h in culture,
[
H]palmitate (60 µCi/dish) was present in the
dish. Cells were washed once with 10 ml of PBS per dish and cultured in
MEM containing 100 µg/ml BSA for 2 h at 37 °C. A,
cells were either not treated (
) or incubated in the presence
of IL-1
(10 ng/ml) for 5 min (
). B, cells were
either not treated (
) or incubated for 5 min in the presence of
IL-1
plus 50 µg/ml D609 (
). C, cells were
either not treated (
) or incubated in the presence of 20
µg/ml DAG C8:0 for 5 min (
). Cells were washed with ice-cold
PBS and fractionated on sucrose gradients. Lipids from each fraction
were extracted and separated by TLC. The ceramide spot was scraped and
counted.
We have localized IL-1-mediated ceramide production to a
highly select region of membrane that has the characteristics of
caveolae. Arachidonate labeling indicates that this fraction represents
1% of the total membrane, yet it contains all of the detectable
caveolin, most of the GPI-anchored alkaline phosphatase, most of the
plasma membrane PKC
(12) , and is enriched in
cholesterol(8) . All of these molecules have been shown by
morphologic methods to be concentrated in invaginated caveolae. This
fraction of membrane also contains 50-70% of the sphingomyelin in
the cell. Nearly all of this sphingomyelin was at the cell surface. The
high content of sphingomyelin and cholesterol most likely accounts for
the Triton X-100 insolubility of the lipid and GPI-anchored protein
components of this membrane(42) .
Hormones that stimulate
ceramide production utilize either a neutral or an acid
sphingomyelinase(40) . Our results suggest that in human
fibroblasts, IL-1 uses an acidic enzyme to generate ceramide.
First, we were able to detect an acid-dependent sphingomyelinase in the
caveolae fractions (Fig. 8). Most likely, this enzyme requires a
low pH compartment to be active. Second, plasmalemmal vesicles appear
to be an acidic compartment(7, 43) . Finally, we found
that IL-1
-stimulated ceramide production in human fibroblasts is
strictly dependent on DAG formation (Fig. 9). Previous work has
shown DAG to be an intermediate specific for the acid sphingomyelinase
pathway (29) .
Besides offering a suitable environment for
ceramide production, caveolae provide a way to deliver this molecule to
other compartments in the cell. Smart et al.(8) have
shown that oxidation of membrane cholesterol causes caveolin to move
from the plasma membrane to the Golgi apparatus by way of the ER. This
appears to be a normal cycle ()required for shuttling
sterols between the two compartments. Since caveolin is a high affinity
binding site for long chain, unsaturated fatty acids (45) , (
)it may also transport fatty acids to the ER. This would
explain why caveolin (47) and caveolae (48) dramatically increase during adipocyte differentiation.
Therefore, IL-1
binding may be coupled to caveolae-mediated
transport of the ceramide to the ER, where subsequent steps in the
signal cascade take place.
The potential transport of cholesterol to the ER by caveolae also suggests a reason for why ceramide formation can stimulate cholesterol esterification(46, 49, 50) . This occurs either after the addition of sphingomyelinase to the cell (49, 50) or when endogenous sphingomyelinase is activated(46) . Cholesterol esters are synthesized by the ER enzyme, acyl coenzyme-A cholesterol acyltransferase. Acyl coenzyme-A cholesterol acyltransferase is constitutively active (44) and siphons off any excess ER cholesterol for storage in lipid droplets. Stimulation of ceramide production in caveolae may mobilize the cholesterol normally complexed with sphingomyelin. This cholesterol would then be available for transport to the ER by caveolae. When the ER cholesterol pool rises too high, acyl coenzyme-A cholesterol acyltransferase diverts the excess into storage.
IL-1-dependent
ceramide production in fibroblasts is an example of a specific
signaling molecule that is made in caveolae in response to a hormonal
stimulus(28) . All of the ceramide produced in the cell
appeared in the caveolae fraction (compare Fig. 2B with Fig. 6), even though IL-1
stimulated an increase in DAG in
both the caveolae and the whole cell fractions (Fig. 8). More
remarkably, the addition of synthetic DAG C8:0 to the media only
stimulated ceramide production in caveolae (Fig. 9C).
What caveolae must do is spatially segregate sphingomyelin on the cell
surface into a compartment that is optimally responsive to one type of
stimulus. This ensures that the newly made ceramide will only go to a
location in the cell where it can act on a specific signaling pathway.
Cells that use ceramide for more than one signaling activity must have
multiple pools of sphingomyelin(29) .