(Received for publication, May 26, 1995; and in revised form, July 21, 1995)
From the
Sphingosine is a product of sphingolipid metabolism that has
been linked to a protein kinase C-independent mitogenic response. In
previously published data, utilizing an in vitro model system
for platelet-derived growth factor (PDGF)-induced vascular smooth
muscle proliferation, we have demonstrated that sphingosine is
increased at the expense of a concomitant decrease in ceramide
formation, implicating an altered ceramidase activity. To explore
mechanisms of growth factor-stimulated sphingosine formation, we have
developed and investigated a cell-free model system assessing
ceramidase activity. We now report that an alkaline,
membrane-associated, ceramidase activity in the rat glomerular
mesangial cell, a smooth muscle-like pericyte, is up-regulated by
growth factors, apparently via a tyrosine kinase phosphorylation
mechanism. PDGF also stimulated sphingomyelinase activity which
generates sufficient substrate to drive the subsequent ceramidase
reaction. Inflammatory cytokines, including interleukin-1, and tumor
necrosis factor-, stimulated sphingomyelinase but not ceramidase
activity, a result consistent with the cellular accumulation of the
ceramide, apoptidic, differentiating second messenger. Mitogenic
vasoconstrictor peptides such as endothelin-1 stimulated neither
sphingomyelinase nor ceramidase activities. An inhibitor of ceramidase
activity, N-oleoylethanolamine, reduced PDGF- but not
endothelin-1-stimulated proliferation. Thus, we conclude that, in
mesangial cells, growth factors but not vasoconstrictor peptides or
cytokines induce mitogenesis, in part, through ceramidase-mediated
sphingosine formation.
Sphingolipids are complex ubiquitous lipids that have been
relegated to serving a structural role in membranes. Sphingolipids are
characterized as consisting of a long chain amino dialcohol base
(sphingoid), an amide-linked fatty acyl group, and a polar or
glycosidic head group. Over the last few years, sphingolipid
derivatives have been identified as endogenous membrane
signal-transducing molecules. Sphingomyelin, the major membrane
sphingolipid, can be hydrolyzed by sphingomyelinase to form ceramide, a
second messenger which stimulates differentiation, inhibits
proliferation, and has been associated with
apoptosis(1, 2) . Several cytokines and steroids have
been shown to stimulate sphingomyelinase and form ceramide. Tumor
necrosis factor- (TNF-
), (
)interleukin-1
,
-interferon, and 1
,25-dihydroxy vitamin D
activate sphingomyelinase in hematopoietic cell
lines(3, 4, 5) . The sphingomyelin signaling
pathway stimulated by TNF-
and interleukin-1
can be
reconstituted in cell-free extracts (4, 6) and may be
mediated by arachidonic acid(7) . Ceramides are themselves
substrates for ceramidases that form the promitogenic lipid,
sphingosine(8) . Our laboratory and the laboratory of Spiegel
have recently reported that platelet-derived growth factor (PDGF)
stimulates mitogenic, sphingolipid-derived, second messengers including
sphingosine and sphingosine-1-phosphate by degrading
ceramide(9, 10) . These data are consistent with the
hypothesis that growth factors, but not inflammatory cytokines,
activate alkaline ceramidase, a membrane-associated enzyme that has
only been partially characterized (11) . A synthetic analog of
sphingosine, N-oleoylethanolamine, has been shown to be a
potent inhibitor of ceramidase in vitro(11) .
Utilizing this inhibitor, we investigate the contribution of ceramidase
activation to growth factor-induced proliferation in MC. MC are a
useful model to explore proliferative responses and phenotypic changes
in a contractile, smooth muscle-like cell. Ligands for both tyrosine
kinase-linked receptors (PDGF) and G protein-linked receptors (ET-1)
are potent mitogens for MC(12) . However, it is not completely
understood how the biochemical events initiated by either PDGF- or
ET-1-binding results, within hours, in DNA replication and cell
division. Cytokines, including IL-1
, are, at best, poor
co-mitogens for smooth muscle cell types(13) . In fact,
IL-1
induces a phenotypic change in MC, switching these cells from
a contractile, promitogenic state to a secretory, nonproliferating
phenotype(13) . Thus, PDGF- or ET-1-treated MC can be used as in vitro models for a myogenic, mitogenic, phenotypic state
while IL-1
-treated MC can serve as an in vitro model for
an inflammatory, secretory phenotypic state. In the present study, we
correlate sphingomyelinase-activated ceramide formation and
ceramidase-stimulated sphingosine generation with growth factor-,
vasoconstrictor peptide-, and cytokine-induced cellular proliferation
and differentiation.
Growth factors and cytokines were purchased from Upstate
Biotechnology, Inc. (Lake Placid, NY) and ET-1 was purchased from
Peptide Institute (Tokyo, Japan). Genistein and all required cell
culture media were obtained from Life Technologies, Inc.
[H]Thymidine was obtained from DuPont NEN. All
other reagents were obtained from Sigma. Membrane, cytosolic, and total
cellular protein content from MC was determined by the method of Lowry
(Kester(14) ).
We and others have previously demonstrated that PDGF
stimulates [H]sphingosine at the expense of
[
H]ceramide and that inhibition of sphingolipid
metabolism correlates with a reduction in PDGF-induced
mitogenesis(9, 10) . We have previously verified in MC
and in A
r5 smooth muscle cells that treatment with either
10 ng/ml PDGF-AB or PDGF-BB for 1 h induces maximal elevations in
[
H]sphingosine formation(9) . To better
understand the mechanism of growth factor-stimulated sphingosine
formation, we have developed a cell-free ceramidase assay for MC.
Ceramidase activity was assessed as TLC-separated
[
C]oleate released from PDGF-treated or
untreated membrane preparations and exogenous
[
C]oleoylceramide substrate. In control
experiments (data not shown), PDGF did not significantly stimulate
ceramidase activity in heat (100 °C)-activated or calcium-free
membrane preparations. Also, stimulated ceramidase activity was
observed only when PDGF was added to intact cells and not when PDGF was
incubated with the membrane preparation, suggesting that the PDGF
responses require an intact receptor-linked signaling cascade. MC were
treated with PDGF-BB (10 ng/ml, 1 h) or vehicle in the presence or
absence of the tyrosine phosphatase inhibitor, vanadate (250
µM, 15 min preincubation) at pH 7.4, and then cell-free
ceramidase activity was assessed at three distinct pH values (Fig. 1). PDGF-BB (and in data not shown, PDGF-AB) stimulates
ceramidase activity at alkaline but not at acidic or neutral pH values,
a finding consistent with activation of a plasma membrane-associated
and not an endosomal/lysosomal ceramidase. PDGF-BB activation of
alkaline ceramidase was maximal at 1 h and persisted for up to 6 h
(data not shown). The mitogenic vasoconstrictor peptide, ET-1
(10
M, 1 h), did not induce an elevation in
membrane-associated ceramidase activity in the absence or presence of
vanadate. PDGF-stimulated membrane ceramidase activity was, however,
further augmented in the presence of vanadate, suggesting that maximal
activity is associated with a tyrosine phosphorylation mechanism.
PDGF-stimulated ceramidase activity was observed at a neutral pH value
in the presence but not the absence of vanadate, suggesting that, under
optimal assay conditions, growth factors can stimulate ceramidase
activity at physiological pH values.
Figure 1: PDGF, but not ET-1, stimulates membrane-associated alkaline ceramidase activity in MC. n = 4, each n replicated in duplicate or triplicate, mean ± S.E., p < 0.05.
A definitive role for tyrosine
phosphorylation regulation of sphingosine formation was further
suggested as PDGF-stimulated ceramidase activity was inhibited in the
presence of the tyrosine kinase inhibitor, genistein (25
µM, 1-h preincubation). Genistein significantly inhibited
PDGF-BB- but not vehicle-treated MC ceramidase activity (Fig. 2). These experiments were run in the presence of vanadate
to ensure maximal phosphorylation and/or activity of ceramidase. Also,
these MC data were confirmed in Ar5 vascular smooth muscle
cells (data not shown) as to demonstrate that PDGF-stimulated
ceramidase activity is not unique to a smooth muscle-like pericyte.
Figure 2: PDGF stimulates MC ceramidase through a tyrosine kinase phosphorylation mechanism. PDGF-stimulated membrane-associated ceramidase activity was inhibited in the presence of the tyrosine kinase inhibitor, genistein. n = 3 separate experiments, each n replicated in duplicate or triplicate, mean ± S.E., p < 0.05.
We next assessed the relative specificity for ceramidase activation
by growth factors (Fig. 3). Even though PDGF-BB was the most
potent growth factor, basic fibroblast growth factor, insulin-like
growth factor, and epidermal growth factor (all 10 ng/ml, 1 h) still
significantly stimulated ceramidase activity. In contrast, the
inflammatory cytokines, IL-1 and TNF-
, did not stimulate
ceramidase activity even in the presence of vanadate. Thus, growth
factors, but not cytokines or mitogenic vasoconstrictor proteins,
stimulate ceramidase activity in vitro.
Figure 3: Growth factors but not cytokines stimulate MC ceramidase activity. MC were treated with all agonists (10 ng/ml) for 1 h and membrane ceramidase activity was assessed. n = 4 separate experiments, each n replicated in triplicate, mean ± S.E., p < 0.05.
To assess if
sufficient substrate could be generated in vivo for optimal
ceramidase activity, we measured MC sphingomyelinase activity, in a
cell-free system, as an indicator of ceramide formation (Fig. 4). PDGF-BB (10 ng/ml, 1 h) stimulates sphingomyelinase
activity which generates sufficient substrate for subsequent ceramidase
activity. Confirming reports by other
investigators(5, 7) , IL-1 and TNF-
(both at
10 ng/ml) were potent activators of sphingomyelinase activity in MC.
However, these cytokine-receptor-induced signals were incapable of
stimulating ceramidase activity. Thus, these data are consistent with
cytokines as inducers of ceramides and a resulting differentiated,
nonproliferative, cellular phenotype. ET-1 (10
M) neither stimulated sphingomyelinase nor ceramidase
activities, suggesting that the mitogenic actions of this
vasoconstrictor are not mediated by sphingolipid metabolites.
Figure 4: PDGF and cytokines stimulate MC sphingomyelinase activity. n = 3 experiments, each n replicated in triplicate, mean ± S.E., p < 0.05.
To
further ascertain the role of sphingolipid metabolites, especially
sphingosine, in mediating growth factor-induced proliferation, we have
utilized an inhibitor of ceramidase, N-oleoylethanolamine (0.5
mM, 16 h dissolved in 5% MeSO, 95% buffer
containing 0.5% bovine serum albumin), in
[
H]thymidine incorporation studies. N-Oleoylethanolamine significantly inhibited PDGF-BB (10
ng/ml)- but not ET-1 (10
M)-stimulated
mitogenesis in MC (Fig. 5). N-Oleoylethanolamine had no
effect by itself on proliferation. Exogenous sphingosine (10
µM) partially restored the mitogenic response to PDGF but
not ET-1. These data imply that the mitogenic actions of PDGF, but not
ET-1, were mediated through sphingosine or a sphingosine derivative.
Moreover, in data not shown, IL-1
or TNF-
are not mitogens
for MC under these assay conditions, supporting our observations that
cytokines do not stimulate ceramidase activity. We conclude that, in
mesenchymal cells, growth factors stimulate ceramidase activity
resulting in the formation of the mitogenic lipid, sphingosine.
Figure 5: Inhibition of ceramidase activity reduces PDGF- but not ET-1-stimulated mitogenesis. n = 4, each n replicated in quadruplicate, mean ± S.E., p < 0.05.
We have extended our earlier studies that implicate sphingosine as a component of PDGF-induced mitogenesis in mesenchymal cells(9) . We now report that growth factors, but not mitogenic vasoconstrictor peptides or inflammatory cytokines, stimulate ceramidase activity in vitro. Both an increase in sphingosine and a decrease in ceramide content is consistent with a mitogenic phenotype. This is the first report to dissociate a role for distinct sphingolipid metabolites in a proliferative versus a differentiated phenotype. By making parallel evaluations of ceramidase and sphingomyelinase activities in vitro, we have demonstrated that even though both PDGF and cytokines induce sphingomyelinase activation, only growth factor-receptors have the capacity to also augment ceramidase activity. These studies offer a novel explanation for the differentiating, antiproliferative, actions of cytokines and the promitogenic actions of growth factors. Consistent with our observations are studies that implicate ceramide in cell cycle arrest and apoptosis (2) and sphingosine metabolites in cellular proliferation(9, 10) .
The role of sphingosine to mediate, in part, the mitogenic actions of growth factors but not of a mitogenic vasoconstrictor peptide is a particularly exciting finding. At the minimum, these observations suggest that the actions of N-oleoylethanolamine are specific to a sphingolipid-mediated signaling pathway. At the maximum, these studies suggest that mitogenic actions of sphingosine are distinct from proliferative signals induced by G protein-linked receptors. Yet, surprisingly, to date, all of the putative mechanisms postulated for the mitogenic actions of sphingosine can be induced by G protein-linked receptors. For example, sphingosine as well as ET-1 have been shown to induce elevations in intracellular free calcium concentrations(17, 18) , activation of phospholipase D resulting in the formation of the mitogens, phosphatidic and lysophosphatidic acids(19, 20) , and DNA binding activity of AP-1 transactivating elements(21, 22) . Thus, sphingosine may regulate cellular proliferation through, as yet, unknown signaling mechanisms. In this regard, phospholipid-derived signaling pathways including phospholipase C, protein kinase C, or c-fos are not required for PDGF-induced proliferation(23, 24) .
As PDGF-induced mitogenesis is, in part, sphingosine-dependent, it is surprising that sphingosine formation mediated by growth factor-regulated sphingolipid metabolism has not been investigated in pathophysiologies that manifest a proliferative smooth muscle phenotype. To date, sphingolipid metabolism and resulting proliferation and/or hypertrophy has only been investigated in the streptozotocin-treated rat model of diabetic nephropathy(25) . In this model, a glucosyl ceramide synthetase inhibitor has been used to demonstrate that an elevation in glucosyl ceramides with a concomitant reduction in ceramides is associated with renal growth(25) . Thus, a decrease in ceramide content, either by an increase in ceramidase, sphingomyelin synthase, or glucosyl ceramide synthetase activities, correlates with a proliferative response. Even though alkaline ceramidase has not been isolated or cloned, a genetic disorder of acidic ceramidase (Farber's lipogranulomatosis) has been identified and linked to accumulation of ceramide and resulting lethal neuronal abnormalities(26) .
In conclusion, we suggest that growth factors, but not inflammatory cytokines or mitogenic vasoconstrictor peptides, mediate proliferation, in part, through ceramidase-regulated sphingosine formation. The dissociation of growth factor-induced mitogenesis from cytokine-mediated differentiation and apoptosis as a consequence of distinct sphingolipid-derived second messengers implicates the sphingomyelin cycle as an important homeostatic regulator of cellular division and differentiation.