(Received for publication, July 23, 1995; and in revised form, August 18, 1995)
From the
Interleukin-1 triggers the down-regulation of several hepatic
cytochrome P450 gene products, but the cellular signaling pathways
involved are not known. We have examined the role of sphingomyelin
hydrolysis to ceramide in the suppression of CYP2C11, a major
constitutive form of cytochrome P450, by interleukin-1. Treatment of
rat hepatocytes cultured on matrigel with interleukin-1 caused a
rapid turnover of sphingomyelin and an increase in cellular ceramide,
with no change in cellular phosphatidylcholine. The ceramide was
composed mainly of a D-erythro-sphingosine backbone,
suggesting that it was derived from sphingolipid hydrolysis rather than
from increased de novo synthesis. Treatment of the cells with
either N-acetyl-D-erythro-sphingosine
(C
-ceramide) or bacterial sphingomyelinase suppressed the
expression of CYP2C11 and induced the expression of the
interleukin-1-responsive
-acid glycoprotein mRNA. In
contrast, the acute-phase gene
-fibrinogen, which is induced by
interleukin-6 but not by interleukin-1, did not respond to
C
-ceramide. N-Acetyl-D-erythro-sphinganine mimicked the
effect of C
-ceramide on CYP2C11, but not on
-acid glycoprotein expression. These results are
consistent with a role for ceramide or a related sphingolipid in
mediating the down-regulation of CYP2C11, the induction of
-acid glycoprotein, and perhaps other cellular effects
of interleukin-1 in hepatocytes.
Stimulation of the immune system during an infection or inflammation results in an impairment of hepatic drug metabolism and a decrease in the hepatic content of cytochrome P450, the family of enzymes that are responsible for the metabolism of many drugs and chemical toxins(2, 3, 4) . The decrease in drug clearance can result in adverse reactions to normal doses of clinically important drugs, such as theophylline(5) , that have low therapeutic indices.
At least part of the decrease in
P450()-catalyzed metabolism in response to inflammatory
stimuli such as bacterial endotoxin is due to the down-regulation of
multiple P450 gene products(6, 7) , which appear to be
manifested mainly at the transcriptional level(8) . The
down-regulation of P450 genes is accompanied by a well characterized
induction of hepatic plasma proteins (such as fibrinogen and
-acid glycoprotein(9) , which are called the
``positive acute-phase proteins'') and a decreased synthesis
of ``negative acute-phase proteins'' (such as
albumin(9) ).
Cytokines, together with glucocorticoids, are
the major humoral mediators of the acute phase of host defense after
injury and infection (9) . Numerous in vivo and in
vitro studies have shown that IL-1 plays a key role in
inducing many of the acute-phase proteins at both transcriptional and
post-transcriptional levels(9) , whereas others are induced
mainly by the action of interleukin-6(9) . A role for IL-1
in the down-regulation of P450 genes has been inferred from several
studies in which injection of IL-1
in vivo reduced the
expression of various P450 gene
products(10, 11, 12, 13) . However,
until now, studies on the mechanism of the IL-1
suppression of
P450 gene expression have been impeded by the lack of a culture system
in which hepatocyte P450s are expressed stably and constitutively at
levels approaching those in vivo and a relative paucity of
knowledge of the intracellular signaling pathways utilized by the
IL-1
receptor to regulate gene expression.
Significant
developments have occurred recently in both of these areas. (a) Hepatocytes cultured on matrigel with insulin as the only
hormone present were shown to express CYP2C11, a major constitutively
expressed P450 in male adult rats(6) , stably and at levels
approaching those seen in vivo in the absence of growth
hormone(14) . We have shown that CYP2C11 expression in this
system is pretranslationally suppressed by IL-1(15) . (b) IL-1
has been found to induce the hydrolysis of
sphingomyelin and accumulation of ceramide in a number of nonhepatic
cell types(16, 17, 18) . Ceramide appears to
play a role as a second messenger since cell-permeable ceramide analogs
and addition of exogenous sphingomyelinase to release endogenous
ceramide can bypass receptor activation and mimic the effect of
TNF-
(19) and IL-1
, e.g. IL-1-mediated
induction of cyclooxygenase mRNA (20) in fibroblasts and
activation of IL-2 production in thymoma cells(21) . This novel
signaling pathway, referred to as the ``sphingomyelin
cycle,'' was originally described in HL-60 cells upon stimulation
with 1
,25-dihydroxyvitamin D
,
-interferon, or
TNF-
(16, 17, 22) . Ceramide has been
implicated as a modulator of a variety of downstream events, such as
activation of specific protein phosphatases and protein kinases,
down-regulation of the c-myc proto-oncogene, apoptosis, and
activation of nuclear factor
NF-
B(16, 17, 18) .
These recent
findings prompted us to investigate whether sphingolipids are involved
in the regulation of P450 and acute-phase genes during inflammation. In
this study, we report that IL-1 initiates SM turnover in
hepatocytes and that exogenous and endogenous ceramides mimic the
effects of IL-1
on the expression of the CYP2C11 and AGP genes.
These results suggest that sphingolipids are involved in the response
of hepatocytes to this cytokine.
Hepatocytes were isolated from
ether-anesthetized, male Sprague-Dawley rats by in situ collagenase perfusion(14) . Cells (3.5
10
/plate; viability > 80%) were plated in 3 ml of
Waymouth's medium containing insulin (0.15 µM) as
the only hormone. Cultures were maintained for 5 days at 37 °C in
5% CO
atmosphere. The medium was replaced every 48 h,
commencing 3 h after plating. In some cases, the hepatocytes were
labeled with [
C]choline chloride (0.4
µCi/dish). To obtain greater labeling of SM, these cells were
cultured in Waymouth's MD 70511 medium, which lacks choline
chloride.
To quantitate the mass of ceramide, 600 pmol of N-acetyl-C-sphinganine/sample (27) was
added to the regions where ceramide migrated (as an internal standard).
These spots were scraped, and 2 ml of chloroform/methanol (1:2, by
volume) was added to each sample. After mixing for 1-2 min, the
silica was sedimented by centrifugation for 10 min in a tabletop
centrifuge. This procedure was repeated three times, and pooled
supernatants were evaporated under reduced pressure. Ceramide mass was
quantitated by analyzing the long-chain bases released after acid
hydrolysis using a reverse-phase HPLC method, and the values were
corrected for recovery of the internal standard.
Figure 1:
Time course of SM degradation upon
treatment with IL-1. Hepatocytes were cultured for 3 days in
modified Waymouth's medium lacking choline chloride, prelabeled
with 0.4 mCi of [
C]choline chloride/dish for
another 48 h, and then treated with 5 mg/ml IL-1
for the indicated
times. Values are means ± S.E. (n =
3).
The IL-1 dose dependences for the loss of radiolabel and mass from
SM are shown in Fig. 2. SM turnover was evident at 1 ng/ml
IL-1 and only increased slightly at higher concentrations.
Suppression of CYP2C11 by IL-1
in rat hepatocytes begins to be
significant at concentrations as low as 0.5 ng/ml and reaches a plateau
at 2 ng/ml(15) . Therefore, the levels of IL-1
that cause
suppression of CYP2C11 also induce SM hydrolysis.
Figure 2:
Effect
of IL-1 on degradation of sphingomyelin. Hepatocytes were cultured
in modified Waymouth's medium and prelabeled with
[
C]choline chloride for 48 h as described for Fig. 1(upper panel) or in Waymouth's medium only (lower panel) and then treated with different concentrations
of IL-1
for 45 min. The upper panel shows the
disappearance of radiolabeled SM, while the lower panel presents changes in its mass, as determined by phosphate assay.
Values are means ± S.E. (n =
3).
Figure 3:
Effect
of IL-1 on hepatocyte ceramide level. Hepatocytes were cultured in
Waymouth's medium for 5 days and treated with the indicated
amounts of IL-1
for 45 min. The ceramide mass was measured by HPLC
of o-phthalaldehyde derivatives of sphingosine, which is
formed after acid hydrolysis of ceramide. Values are means ±
S.E. (n = 3).
Figure 4:
Effect of C-ceramide and
exogenous sphingomyelinase on expression of CYP2C11 mRNA. On day 5 of
culture, hepatocytes were treated with 30 µM C
-ceramide or 0.1 unit/ml bacterial sphingomyelinase (SMase) for the indicated times. Values are means ±
S.E. (n = 5). The value for the 4-h control group is
set arbitrarily to 100%. *, significantly different from the
corresponding control group mean (p <
0.05).
The decreases in CYP2C11 mRNA evoked by
sphingomyelinase and C-ceramide were followed by
suppression of its protein product at 48 and 72 h of incubation (Fig. 5), with a maximal reduction of
65% (CYP2C11 protein
levels remained relatively constant in untreated cultures). Smaller
effects on CYP2C11 apoprotein were observed after 24 h (data not
shown). The effects of sphingomyelinase and C
-ceramide on
CYP2C11 protein were similar in time dependence and magnitude to those
of IL-1 (Fig. 5).
Figure 5:
Reduction in CYP2C11 apoprotein level by
ceramide, exogenous sphingomyelinase, and IL-1. Hepatocytes were
treated with 30 µM C
-ceramide, 0.1 unit/ml
bacterial sphingomyelinase (SMase), or 5 ng/ml IL-1
for
the indicated periods of time. Microsomal protein pooled from two
dishes (5 mg) was subjected to 7.5% SDS-polyacrylamide gel
electrophoresis, transferred to nitrocellulose paper, and probed with
polyclonal antibody for CYP2C11. The blots shown are of representative
samples from each treatment group. The numerical values are the means
± S.E. obtained from densitometry of three independent samples
from each group, expressed as a percentage of the control (zero time)
group mean.
Figure 6:
Dose-dependent effect of
C-ceramide on expression of CYP2C11 (upper panel)
and AGP and
-fibrinogen (lower panel) mRNAs. On day 5 of
culture, hepatocytes were treated with the indicated amounts of
ceramide for 24 h. Values are the means ± S.E. (n = 5).
Figure 7:
Comparative potencies of
C-dihydroceramide and C
-ceramide in regulation
of CYP2C11 and AGP mRNAs. 5-Day cultures of hepatocytes were incubated
with the indicated concentrations of C
-ceramide (
) or
dihydroceramide (
) for 24 h. Values are the means ± S.E. (n = 5-10).
This study provides the first evidence that sphingolipids may
be involved in the regulation of hepatic P450 mRNA by cytokines and
also suggests a role for sphingolipids in the induction of acute-phase
protein gene expression. The induction of SM turnover and ceramide
accumulation by IL-1 implies that ceramides mediate the action of
this cytokine (as has been seen in other systems), and this was
supported by the ability of C
-ceramide to effect the
inflammatory response of two genes known to be regulated by IL-1,
CYP2C11 and AGP, but not the expression of another acute-phase gene,
fibrinogen, which is specifically regulated by interleukin-6.
Furthermore, an exogenously added sphingomyelinase also mimicked the
action of IL-1
in suppression of CYP2C11 expression.
The
finding that ceramide can mimic these effects of IL-1 will allow
experiments to determine if the protein kinase(s) (34) and
phosphoprotein phosphatase(s) (35) that have been proposed as
the target of ceramides in other systems are involved in signal
transduction from IL-1
and related cytokines in hepatocytes. IL-1
regulation of acute-phase genes, including AGP, involves the
participation of at least two types of cis-acting elements on
responsive genes that bind transcription factors of the NF-
B and
cAMP response element-binding protein (C/EBP)
families(36, 37, 38) . Ceramide has been
reported to mediate the activation of NF-
B by TNF-
in HL-60
cells (18, 39) and to activate proteolytic degradation
of I
B in lysates of monocytic cells(40) , but other
workers have found no activation of NF-
B in HL-60 cells or Jurkat
T cells(41, 42) . It remains to be seen whether the
ceramide pathway is coupled to CYP2C11 and/or AGP gene regulation via
NF-
B in our system. However, three observations indicate that the
signaling pathways for regulation of the P450 and type I acute-phase
genes may diverge at a point after ceramide formation. (i) The genes
are affected in an inverse manner by cytokines and ceramide; (ii)
ceramide is more potent in CYP2C11 down-regulation than in AGP
induction; and (iii) C
-dihydroceramide suppresses CYP2C11
expression similarly to C
-ceramide, but
C
-ceramide is more effective in AGP induction than is
dihydroceramide.
The last point is consistent with the participation
of ceramide-activated protein phosphatase in AGP induction, but
suggests that a different cellular target may be involved in the
sphingolipid-mediated down-regulation of P450. One possibility is the
previously described ceramide-sensitive kinase, which is generally more
promiscuous than ceramide-activated protein phosphatase with respect to
the sphingoid molecules that activate it(33) . However, it
should be noted that the lack of a differential response to exogenous
C-ceramides and C
-dihydroceramides in whole
cells does not rule out the participation of ceramide-activated protein
phosphatase in the signaling pathway because we have shown that
addition of C
-dihydroceramide can increase cellular levels
of endogenous free ceramides.
It is also intriguing that the
concentration dependence of the increase in endogenous ceramide by
IL-1 (Fig. 3) is similar to that for AGP induction, but
less similar to that for CYP2C11 suppression(15) , whereas SM
turnover is induced by the low concentrations of IL-1
that
suppress CYP2C11. These observations suggest that additional
sphingolipid metabolites (and possibly other sources of ceramide) may
be involved.
Hepatocytes may prove useful in biochemical studies of
sphingolipid cycles. It is not yet known with certainty, for example,
whether there are larger numbers of sphingolipid hydrolases that
participate in signal transduction, if the enzymes are located in the
plasma membrane and/or other cellular compartments, or how these
systems are regulated. In Jurkat cells, activation of an acidic
sphingomyelinase has been shown in lysosomes subsequent to TNF-
stimulation(43) ; in HL-60 cells, cytosol is considered to be
the source of the cytokine-regulated sphingomyelinase
activity(44) . However, in liver, the majority of the SM is in
the plasma membranes, where a neutral sphingomyelinase activity has
been identified and characterized(45, 46) . It has
been suggested that the sphingomyelinase is downstream from activation
of phospholipase A
(47) . The relative ease with
which highly purified plasma membranes may be isolated from liver (and
even membrane subdomains) (48) should provide a model to help
clarify some of these questions.
The liver is a primary target for
cytokines during the host defense reaction to injury and inflammation.
Among the numerous systems affected is the P450 gene superfamily, which
plays a critical role in the oxidative metabolism of drugs, toxins, and
steroids(49) . Decreases in multiple P450 enzymes during
immunostimulatory conditions can have important clinical
consequences(5) . Similarly, the induction of acute-phase
protein synthesis and secretion is thought to be important in the
homeostasis of inflammation(50) . Therefore, our discovery of a
sphingolipid signaling pathway for IL-1 in rat liver may have
implications not only for altered drug responses caused by the
down-regulation of P450 under these conditions, but also for the
development of the systemic response to infection and inflammation. ()
These findings may have broader implications because CYP2C11 expression is also down-regulated by glucocorticoids in vivo(52) and by growth hormone both in vivo and in vitro(14, 53) . The possible involvement of sphingolipid mediators in these effects should be evaluated because dexamethasone has been reported to stimulate SM hydrolysis in fibroblasts(54) .