From the Division of Rheumatology and Immunology and
the ¶ Department of Biochemistry and Molecular Biology, Medical
University of South Carolina and the
Division of General
Internal Medicine, Ralph H. Johnson Veterans Affairs Hospital,
Charleston, South Carolina 29425
Received for publication, November 12, 2002
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ABSTRACT |
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Transforming growth factor- Transforming growth factor- Sphingolipids, in addition to their role as structural molecules of the
plasma membrane, are now recognized as important bioactive mediators of
a variety of cellular processes (10-12). In particular, biologic
functions of ceramide, sphingosine, and their phosphates have been
extensively studied. Intracellular ceramide is primarily generated via
hydrolysis of sphingomyelin by a family of sphingomyelinases. In
addition, ceramide can be formed from sphingosine by ceramide synthase.
Sphingosine can be further converted to sphingosine 1-phosphate (S1P)
by the action of sphingosine kinase (SPHK1). On the other hand, S1P
phosphatase dephosphorylates S1P back to sphingosine (Fig.
1). The yeast S1P phosphatase, YSR2
(yeast sphingosine resistance gene), which has a high degree of
specificity, has recently been cloned and characterized (13). It has
been shown that overexpression of YSR2 in yeast increased ceramide
formation. Conversely, deletion of YSR2 decreased ceramide levels (14). The biosynthetic pathways of sphingoid bases, their phosphates, and
ceramide are conserved for the most part from yeast to mammals. It was
shown that mammalian S1P phosphatase was able to substitute for YSR2
when expressed in mutant yeast lacking YSR2 function (15). Thus, it has
been proposed that S1P phosphatase may play an important role in
altering the balance between ceramide and intracellular S1P.
Significantly, ceramide and S1P appear to have antagonistic functions,
particularly in the regulation of cell growth. Ceramide, which is
induced by inflammatory cytokines, various damaging agents, and stress
signals, seems to be mainly associated with cell growth arrest and
apoptosis, whereas intracellular S1P has been shown to promote cell
proliferation (10, 11). It is important to note that the effects of
ceramide and S1P on cell growth and other cellular functions are cell
type-specific.
(TGF-
) is a
multifunctional growth factor that plays a critical role in tissue
repair and fibrosis. Sphingolipid signaling has been shown to regulate
a variety of cellular processes and has been implicated in collagen
gene regulation. The present study was undertaken to determine whether
endogenous sphingolipids are involved in the TGF-
signaling pathway.
TGF-
treatment induced endogenous ceramide levels in a
time-dependent manner within 5-15 min of cell stimulation.
Using human fibroblasts transfected with a
2(I) collagen
promoter/reporter gene construct (COL1A2),
C6-ceramide (10 µM) exerted a
stimulatory effect on basal and TGF-
-induced activity of this
promoter. Next, to define the effects of endogenous
sphingolipids on TGF-
signaling we employed ectopic expression of
enzymes involved in sphingolipid metabolism. Sphingosine 1-phosphate
phosphatase (YSR2) stimulated basal COL1A2 promoter activity and
cooperated with TGF-
in activation of this promoter. Furthermore,
overexpression of YSR2 resulted in the pronounced increase of COL1A1
and COL1A2 mRNA levels. Conversely, overexpression of sphingosine
kinase (SPHK1) inhibited basal and TGF-
-stimulated COL1A2 promoter
activity. These results suggest that endogenous ceramide, but not
sphingosine or sphingosine 1-phosphate, is a positive regulator of
collagen gene expression. Mechanistically, we demonstrate that Smad3 is
a target of YSR2. TGF-
-induced Smad3 phosphorylation was elevated in
the presence of YSR2. Cotransfection of YSR2 with wild-type Smad3, but
not with the phosphorylation-deficient mutant of Smad3 (Smad3A),
resulted in a dramatic increase of COL1A2 promoter activity. In
conclusion, this study demonstrates a direct role for the endogenous
sphingolipid mediators in regulating the TGF-
signaling pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(TGF-
)1 is a member of
a large family of growth factors with diverse functions in
embryonic and adult tissues (1). TGF-
plays a critical role in
regulating immune cell function, epithelial cell growth, and
extracellular matrix deposition (2). Abnormal TGF-
signaling has
been implicated in the pathogenesis of a number of diseases, including
cancer and fibrosis (3). TGF-
signals through a heteromeric receptor complex of type I and type II receptor serine-threonine kinases. Intracellular signal transducers termed Smads transmit signals from the
receptor to the nucleus. The members of the Smad family have been
divided into three functional subgroups: receptor-regulated Smads
(R-Smads), the common mediator Smads (co-Smads), and inhibitory Smads
(I-Smads). R-Smads (Smad2 or -3) are phosphorylated on a conserved
SSXS motif by the activated type I receptor leading to their dissociation from the receptor. Subsequently, they form heterocomplexes with co-Smads and upon translocation into the nucleus
contribute to transcriptional regulation of the target genes (1). A
Smad-interacting membrane-bound protein SARA (Smad anchor for receptor
activation) facilitates binding of the Smad2/3 to the activated type I
receptor (4). Recent studies suggest that interaction of SARA with
Smads occurs in early endosomes (5, 6). Another endosomal FYVE domain
protein, Hgs, has also been shown to cooperate with SARA in recruiting
Smad2 and Smad3 to the activin receptor complex (7). Interestingly,
translocation of Smads to the nucleus can be blocked by phosphorylation
of the linker region of R-Smads by Ras/extracellular signal-regulated kinases (ERKs) (8), suggesting that Smads can be directly targeted by
other signaling pathways. There is also increasing evidence for
cross-talk between TGF-
and other signaling pathways (9).
View larger version (30K):
[in a new window]
Fig. 1.
Schematic view of sphingolipid
metabolism. SM, sphingomyelin; SMase,
sphingomyelinase; S1P, sphingosine 1-phosphate;
GlcCer, glucosylceramide.
Previous studies have suggested that ceramide may also be involved in
regulation of collagen metabolism. It was shown that C2-ceramide and sphingomyelinase inhibit collagen gene
expression in hepatic stellate cells and dermal fibroblasts (16, 17). Furthermore, C2-ceramide suppressed 2(I) collagen
(COL1A2) promoter activity (16). Because elevated collagen production
is associated with fibrotic diseases and TGF-
is a primary inducer
of collagen, this study was undertaken to examine the possible role of
sphingolipids in TGF-
regulation of collagen production. Using
exogenous S1P and C6-ceramide and enzymatic manipulation of
endogenous sphingolipids, we demonstrate that intracellular
sphingolipids modulate the TGF-
signaling pathway. Furthermore, we
provide evidence that the effects of sphingolipids are
Smad3-dependent. This study establishes for the first time
direct functional interaction between TGF-
and sphingolipid
signaling pathways.
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MATERIALS AND METHODS |
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Cell Cultures-- Foreskin fibroblast cultures were established from newborn foreskins obtained from delivery suites of local hospitals. Tissue was dissociated enzymatically by 0.25% collagenase type I (Sigma) and 0.05% DNase (Sigma) in DMEM with 10% fetal bovine serum. COS, Mv1Lu, and HEK 293 cells were obtained from the American Type Culture Collection. Cells were grown in DMEM supplemented with 10% fetal bovine serum.
Plasmid Constructs--
The COL1A2-luciferase construct contains
sequences from 353 to +58 bp of the human COL1A2 promoter (18) fused
to the luciferase reporter gene (pGL2 basic, Promega). Cloning of YSR2
has been described previously (13, 14). The open reading frame of YSR2 was recloned into KpnI and EcoR1 sites of
pcDNA3.1 (Invitrogen). The EGFP coding sequence was amplified from
pEGFP-N1 (Clontech, Palo Alto, CA) and inserted
into HindIII and KpnI sites upstream of YSR2.
p3TP-Lux was a gift from J. Massagué (Memorial Sloan-Kettering Cancer Center, New York, NY). The expression construct for the constitutively active form of TGF-
type I receptor (HA-T
RI(TD)) was provided by K. Miyazono (University of Tokyo). Expression vectors
for Smad3 and Smad3A, which carry three carboxyl-terminal serine-to-alanine substitutions, were gifts from H. Lodish (Whitehead Institute, MA). SPHK1a was subcloned as described by Olivera et al. (19) into pcDNA3.1 vector.
Adenoviral Constructs-- Adenoviral vectors expressing YSR2 and green fluorescent protein (GFP) were generated using a published protocol (20). Briefly, the cDNA encoding YSR2 was cloned into the shuttle vector pAdTRACK-CMV, which contains a GFP expression cassette driven by a separate CMV promoter. The shuttle vector containing YSR2 was cotransformed into Escherichia coli BJ5183 cells with the AdEasy-1 adenoviral backbone plasmid, which lacks the E1 and E3 regions of the adenoviral genome. Linearized recombinant plasmid DNA was transfected into 293 cells, an adenoviral packaging cell line, using the FuGENE 6 transfection reagent (Roche Molecular Biochemicals) to generate the recombinant adenovirus expressing YSR2 and GFP from separate CMV promoters (AdYSR2). An adenovirus expressing GFP alone (AdGFP) was generated via the same method for use as a control vector.
Transient Transfection and Luciferase Assay--
For transient
transfection, 60-80% confluent cells in 6-well plates were
transfected using FuGENE 6 transfection reagents (Roche Molecular
Biochemicals) following the manufacturer's recommendations. After
transfection, cells were incubated for 20 h and stimulated where
indicated for an additional 24 h with TGF- (R & D Systems, Minneapolis, MN), C6-ceramide, S1P, or sphingosine
(synthesized as described in Ref. 21). Luciferase activities in
the cell lysates were measured with a dual luciferase reporter assay
system (Promega).
RNA Preparation and Northern Blot Analysis--
Human
fibroblasts (5 × 104) were plated in 10% fetal calf
serum/DMEM in 6-well plates. Next day, cells were transfected using FuGENE 6 reagent with either empty vector or YSR2. 20 h after transfection, the number of GFP-positive cells was visually determined using a Zeiss Axiovert 35 microscope. Efficiency of transfection in
dermal fibroblasts varied from 5 to 15%. Experiments in which efficiency of transfection was below 10% were not continued. Medium was changed to 1% fetal bovine serum/DMEM, and TGF-1 (5 ng/ml) was
added for an additional 24 h. Total RNA was extracted and analyzed
by Northern blot as described previously (22). Filters were
sequentially hybridized with radioactive probes for COL1A1, COL1A2, and
18 S ribosomal RNA. The filters were scanned with a PhosphorImager
(Amersham Biosciences).
Immunoprecipitation and Immunoblotting--
For determination of
phosphorylation levels of ectopically expressed Smad3, cell lysates
were subjected to immunoprecipitation with the anti-FLAG antibody
followed by adsorption to protein G-Sepharose (Amersham Biosciences).
Precipitates were separated by SDS-PAGE. After immunoblotting
with the anti-phosphoserine antibody (Zymed Laboratories
Inc., South San Francisco, CA), the membranes were subjected to
re-blotting with anti-FLAG (M2, Sigma) antibody to confirm levels of
expression of the FLAG-tagged Smad3 protein. To determine levels of
expression of the receptors, aliquots of cell lysates were subjected to
SDS-PAGE and immunoblotting with anti-HA (Santa Cruz Biotechnology,
Santa Cruz, CA). To determine the kinetics of phosphorylation of
endogenous Smad3, Mv1Lu cells or dermal fibroblasts were treated with
TGF-1 (2.5 ng/ml) for various time intervals. Cell lysates were
subjected to immunoprecipitation with the anti-Smad2/3 antibody (N-19,
Santa Cruz Biotechnology) followed by adsorption to protein
G-Sepharose. Western blotting was performed using an anti-phosphoserine
antibody. As loading control, membranes were subjected to re-blotting
using an antibody against Smad3 (FL-425, Santa Cruz Biotechnology).
Assay of Sphingosine Kinase Activity--
Foreskin fibroblasts
were transiently transfected with either SPHK1a or empty vector. After
a 24-h incubation in DMEM/1% fetal bovine serum, cells were harvested
and lysed in buffer containing 20 mM Tris, pH 7.4, 20%
glycerol, 1 mM -mercaptoethanol, 1 mM EDTA,
1 mM sodium orthovanadate, 40 mM
-glycero-phosphate, 15 mM NaF, 10 µg/ml leupeptin,
aprotinin, and soybean trypsin inhibitor. Sphingosine kinase activity
was determined in the presence of 50 µM sphingosine,
dissolved in 5% Triton X-100 (final concentration 0.25%), and
[32P]ATP (10 µCi, 1 mM) containing
MgCl2 (10 mM) as described in Ref. 23. The
labeled S1P was identified by thin layer chromatography with
1-butanol/ethanol/acetic acid/water (80:20:10:20) and visualized by autoradiography.
Assay of S1P Phosphatase Activity-- Phosphatase activity was measured as previously described (15). Briefly, HEK 293 cells were transfected with either YSR2 or empty vector. Forty-eight hours after transfection, cells were lysed in buffer A (100 mM Hepes, pH 7.5, containing 10 mM EDTA, 1 mM dithiothreitol, and 10 µg/ml each leupeptin, aprotinin, and soybean trypsin inhibitor), and cells were scraped on ice. Cells were subsequently freeze-thawed seven times, then centrifuged at 100,000 × g for 1 h. Supernatants were removed, and the membrane fractions were resuspended in 200 µl of buffer A and homogenized. 32P-labeled S1P was prepared by in vitro phosphorylation of sphingosine using cell lysates obtained from 293 cells overexpressing SPHK1. Alternatively, D-erythro-[4,5-3H]dihydrosphingosine-1-phosphate (ARC, Inc.) was used as a substrate. Similar results were obtained with both substrates. S1P phosphatase activity was measured by adding 32P-labeled S1P to 20 µg of membrane preparations in 200 µl of buffer A and incubated for 30 min at 30 °C. The assay was terminated by adding 20 µl of 1N HCl, and lipids extracted with 0.8 ml of chloroform/methanol/HCl (100:200:1). To separate phases, 0.24 ml of chloroform and 0.24 ml of 2 M KCl were added. After centrifugation, 100 µl of the aqueous layer was counted by liquid scintillation. Remaining 32P-labeled S1P was extracted and analyzed by TLC.
Measurment of Cellular Ceramide--
Mv1Lu cells or dermal
fibroblasts were grown to near confluence, and culture medium was
changed to serum-free DMEM for 24 h, followed by stimulation with
TGF-1 (2.5 ng/ml) for various time intervals. Lipids were then
extracted with chloroform/methanol/water and processed for
determination of ceramide as described (24).
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TGF- Stimulates Endogenous Ceramide Levels--
Modulation of
ceramide levels by a variety of stimuli has been observed in many cell
types; however, only limited information is currently available
regarding TGF-
regulation of ceramide (25). To examine whether
ceramide is involved in TGF-
signaling, we measured ceramide levels
in cells treated with TGF-
for various time intervals. Ceramide
measurements were performed in human dermal fibroblasts and in Mv1Lu
cells. Both cell types responded in a similar manner with a rapid
increase of the ceramide levels in response to TGF-
. As shown in
Fig. 2A, ceramide increased 5 min after TGF-
addition, peaked by 10 min, and very slowly declined
over the next 2 h. We also examined phosphorylation of Smad3 in
cells treated with TGF-
for the same time intervals (Fig.
2B). Detectable phosphorylation of Smad3 occurred at 10 min
after TGF-
addition, with the peak phosphorylation between 20-60
min and a decline at 120 min. These results indicate that TGF-
treatment leads to a relatively rapid induction of endogenous ceramide.
Furthermore, generation of ceramide preceded Smad3 phosphorylation, raising the possibility that ceramide may be involved in TGF-
signaling.
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Exogenous S1P and C6-ceramide Modulate
TGF--dependent COL1A2 Promoter Activity--
Because
our laboratory is particularly interested in defining TGF-
signaling
pathways relevant to fibrosis, we utilized a well characterized COL1A2
promoter reporter construct to delineate the role of ceramide in
TGF-
regulation of matrix genes (18, 22, 26). It has been previously
shown that TGF-
stimulation of this promoter is
Smad3-dependent (27, 28). We first examined the effects of
exogenously added sphingolipids on the basal and TGF-
-dependent COL1A2 promoter activity. Dermal
fibroblasts were transfected with the COL1A2 promoter and treated with
S1P (10-30 µM), C6-ceramide (10-50
µM), and sphingosine (1 µM) in the presence or absence of TGF-
. S1P at micromolar concentrations slightly stimulated basal and TGF-
-induced COL1A2 promoter activity (Fig. 3A), whereas nanomolar doses
of S1P had no effect on the COL1A2 promoter (data not shown).
C6-ceramide (10 µM) stimulated COL1A2 promoter activity with an effect comparable with that of TGF-
, a
known inducer of collagen gene transcription (Fig. 3B).
Simultaneous addition of C6-ceramide (10 µM)
and TGF-
resulted in a further increase of the COL1A2 promoter
activity. On the other hand, C6-ceramide at a higher
concentration (50 µM) did not affect basal promoter levels and decreased TGF-
-induced COL1A2 promoter activity (Fig. 3B). Sphingosine (1 µM) had no effect on the
COL1A2 promoter activity in the presence or absence of TGF-
(Fig.
3C). Higher concentrations of sphingosine were toxic. These
experiments suggest that sphingolipids may be modulators of the basal
and TGF-
-dependent COL1A2 promoter activity in dermal
fibroblasts.
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Interestingly, ceramide had both stimulatory (at lower doses) and inhibitory (at higher doses) effects. One possible interpretation of these results is based on the current view of the dynamic balance between intracellular levels of ceramide, sphingosine, and S1P (10). Because the relative ratio of endogenous ceramide and S1P is tightly controlled, the stimulatory effect of high concentrations of S1P may be because of its conversion to ceramide. Also, the failure of S1P to act in nanomolar range argues against its effects being mediated through the S1P receptors and further supports intracellular action, which would include metabolic interconversion as a distinct possibility. Conversely, inhibitory effects of high doses of ceramide may reflect its metabolism to S1P or other inhibitory intermediates. Together, the experiments using exogenous sphingolipids suggest that balance between components of sphingolipid pathway may be important for collagen gene regulation.
YSR2 and TGF- Synergize to Induce COL1A2 Promoter--
To gain
more understanding into the nature of endogenous sphingolipids involved
in TGF-
signaling, we took advantage of the availability of
recombinant enzymes involved in regulation of the balance between S1P
and ceramide (see Fig. 1). First, we utilized YSR2 to generate
endogenous ceramide. YSR2 specifically dephosphorylates S1P to
sphingosine, which is then converted to ceramide. Control experiments
indicated that cell lysates prepared from cells ectopically expressing
YSR2 contained higher levels of S1P phosphatase activity as compared
with cell lysates obtained from cells expressing empty vector (Fig.
4A). We also determined that
ectopic expression of YSR2 elevates endogenous ceramide levels (Fig.
7D). Overexpression of YSR2 in dermal fibroblasts led to a
modest but consistent induction of the COL1A2 promoter activity.
Significantly, TGF-
stimulation of this promoter was greatly
potentiated by the presence of YSR2 (Fig. 4B). To determine
whether activation by YSR2 is specific for the COL1A2 promoter or
whether YSR2 is a general coactivator of the TGF-
pathway, we used a
TGF-
-responsive artificial reporter system (p3TP-Lux promoter). YSR2
induced basal activity of the p3TP-Lux promoter and synergized with
TGF-
in activation of this promoter, suggesting a general effect of
YSR2 on the TGF-
signaling pathway (Fig. 4C).
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We next examined the effects of overexpressing SPHK1 on COL1A2 promoter
activity. This enzyme generates S1P by phosphorylating sphingosine.
Control experiments indicated that cell lysates prepared from cells
ectopically expressing SHPK1 contained higher levels of SPHK1 activity
(Fig. 5A). Overexpression of
SHPK1 in dermal fibroblasts slightly decreased both basal and
TGF--stimulated COL1A2 promoter activity (Fig. 5B).
Together, these experiments indicate that perturbation of the
intracellular sphingolipid-ceramide pathway has both positive and
negative effects on TGF-
signaling, such that tilting the balance
from S1P to ceramide exerts a pronounced stimulatory effect, whereas
tilting the balance from ceramide S1P induces a modest inhibitory
effect.
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YSR2 Induces Endogenous Collagen Type I Gene Expression--
Next,
we examined the effects of the ectopically expressed YSR2 on expression
levels of COL1A1 and COL1A2 mRNAs in human dermal fibroblasts
transiently transfected with YSR2. Both COL1A1 and COL1A2 mRNAs
were significantly increased in cells transfected with YSR2 as compared
with cells transfected with empty vector (Fig.
6). Depending on the individual cell
line, stimulation of COL1A1 by YSR2 varied from 1.6- to 9.9-fold,
whereas stimulation by TFG- varied from 1.7- to 8.8-fold. When both
stimuli were present, an additional modest increase was observed (2.2- 11.3-fold). It should be noted that in human dermal fibroblasts
autocrine TGF-
signaling pathway is operational and contributes to
basal collagen gene expression (29). Thus, increased COL1A1 and COL1A2 mRNA levels in cells transfected with YSR2 may reflect cooperation between YSR2 and low levels of endogenous TGF-
signaling pathway. Presently, we cannot exclude that other signaling pathways that are
involved in collagen gene regulation are also affected by YSR2.
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YSR2 Regulates Smad3--
Smad2 and Smad3 are mediators of TGF-
signaling. Furthermore, we have previously shown that Smad3 mediates
TGF-
stimulation of the COL1A2 promoter in dermal fibroblasts (28).
Therefore, we next examined the effects of YSR2 on Smad3 stimulation of
the COL1A2 promoter. Transfection with Smad3 or YSR2 alone resulted in
induction of the promoter activity. However, cotransfection of the
COL1A2 promoter with Smad3 and YSR2 led to a dramatic increase in
collagen promoter activity (Fig.
7A). Interestingly,
cotransfection of Smad3A (a mutant of Smad3 that carries three
carboxyl-terminal serine-to-alanine substitutions) (30) also produced a
modest stimulatory effect. This may be because of activation of
endogenous Smad3, or perhaps YSR2 can enhance TGF-
signaling through
an additional yet unidentified mechanism. Because recent studies have
shown that nuclear import of Smad can occur without phosphorylation on
the SSXS motif (31), it is possible that the action of a phosphatase facilitates dissociation of Smad3 from SARA. In any case,
our data suggest that Smad3 is a major effector of YSR2.
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Because phosphorylation of C-terminal serine residues of Smad3 is
necessary for its activation, we investigated the effect of YSR2 on
Smad3 phosphorylation. Using COS cells, Smad3 was coexpressed with the
constitutively active form of the TGF receptor (T
RI(TD)) in the
absence or presence of YSR2. Phosphorylation of Smad3 was detected by
immunoblotting using antibodies to phosphorylated serine residues. As
previously shown, in the presence of T
RI(TD), Smad3 phosphorylation
was observed. YSR2 significantly increased Smad3 phosphorylation (Fig.
7B). This result indicates that YSR2 has an additive or
synergistic effect on the intensity of the TGF-
signaling cascade
acting at the post-receptor level.
To further investigate the role of YSR2 in Smad3 activation, we
ectopically expressed YSR2 in human fibroblasts using adenoviral vector. As shown in Fig. 7C, Smad2/3 was not phosphorylated
in unstimulated cells but became rapidly phosphorylated in response to
TGF- stimulation. In contrast, in cells transduced with YSR2, Smad3
became fully phosphorylated even in the absence of TGF-
with no
additional increases in phosphorylation after addition of TGF-
.
Thus, the endogenous metabolic products of YSR2 (e.g. ceramide) are capable of inducing phosphorylation of Smad3.
Previous studies have shown that overexpression of S1P phosphatase
results in generation of ceramide (13-15). The endogenous levels of
ceramide were measured in cells transduced either with control-GFP or
with YSR2 adenoviruses and compared with cells stimulated with TGF-.
As shown in Fig. 7D, ceramide levels were similarly induced
by YSR2 overexpression or TGF-
treatment.
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DISCUSSION |
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The present study establishes for the first time that endogenous
sphingolipids are co-regulators of the TGF- signaling pathway. The
following observations support this conclusion. First, exogenously added C6-ceramide stimulated basal and TGF-
-induced
collagen promoter activity. Second, overexpression of the yeast S1P
phosphatase had a synergistic effect with TGF-
in activation of the
collagen promoter and stimulated endogenous collagen gene. Both the
yeast and the murine enzymes have been previously shown to decrease endogenous S1P levels and to increase endogenous ceramide levels (14,
15). On the other hand, TGF-
induction of this promoter was
inhibited by overexpression of SPHK1, an enzyme that
promotes formation of S1P. More importantly, TGF-
treatment induced
endogenous ceramide levels, which correlated with TGF-
-induced Smad3
phosphorylation. These results strongly suggest that ceramide is a
coactivator of the TGF-
signaling pathway and that S1P is a possible
endogenous inhibitor. We have also begun to unravel the specific
regulatory mechanisms involved in the interaction of sphingolipids and
TGF-
signaling pathways. We demonstrate by functional assays that
the effects of YSR2 are mediated via Smad3. Consistent with these results, Smad3 phosphorylation is increased in the presence of ectopically expressed YSR2.
Our results also clarify previous observations indicating an inhibitory
role of C2-ceramide in collagen production (16, 17). In our
study the inhibitory effects of exogenous ceramides on COL1A2
transcription were also observed with higher doses of C6-ceramide. These observations suggest that sphingolipids
may have a dual role in collagen gene regulation and that other
metabolites with inhibitory functions may also be formed. S1P may be
one of the metabolites with the inhibitory function. Our recent studies have shown that exogenously added S1P and lysophosphatidic acid inhibit
TGF- stimulation of collagen mRNA stability. These effects were
mediated via MEK/ERK.2
Induction of the ERK pathway was also responsible for collagen inhibition by C2-ceramide (16). Although induction of ERK
pathway in response to exogenously added ceramide or SMase has been
observed in several experimental systems (11), the specific
intracellular mediators of this induction are presently unknown.
C6-ceramide has also been shown to activate p38 pathway
(32). In contrast to ERKs, p38 has been shown to cooperate with TGF-
signaling in collagen gene stimulation (33). However, p38 does not
appear to be involved in the effects observed in our study. First,
Smad3 is not a direct target of p38 (34, 35). Second, stimulation of
the COL1A2 promoter by YSR2 was insensitive to SB203580, a specific
inhibitor of p38 (data not shown). We could also exclude stress-activated protein kinase/c-Jun NH2-terminal kinase
pathway because it was shown that induction of this pathway
down-regulates COL1A2 promoter in dermal fibroblasts (36). Thus, it
appears that the members of the mitogen-activated protein kinase
superfamily, which have been previously shown to mediate many of the
ceramide effects, are not involved in the process described here.
The question remains about the nature of the interaction between the
sphingolipid and TGF- signaling pathways. It has been demonstrated
that both TGF-
receptor type I and type II, which form homodimers in
the endoplasmic reticulum, are clustered in the cell surface into
discrete cellular subdomains (37, 38). Furthermore, a
TGF-
-dependent interaction between T
RI and caveolin-1 has recently been described (39). Interestingly, caveolin-1 was shown
to suppress TGF-
-mediated Smad2 phosphorylation and subsequent
downstream events. It is possible that ceramide may interfere with this
interaction, resulting in increased Smad3 phosphorylation. This
possibility will be tested in our future studies. Recent studies (5, 6)
suggest that assembly of the TGF-
signaling pathway occurs in early
endosomes. Consistent with this possibility, an endosomal protein SARA
has been shown to colocalize with TGF-
receptors and recruit Smad to
the receptor complex (4, 31, 40, 41). Other endosomal proteins,
including Hgs (Hrs), may also contribute to TGF-
signaling (7). It
has been recently shown that ceramide binds to and activates the
endosomal protease, cathepsin D (42). Thus, endosomal ceramide may
facilitate interaction of the TGF-
receptor complex with SARA and
Smad3. Recent studies suggest that the lipid environment may be
critical for the proper interactions of the cell surface receptors with downstream signaling molecules (43). Our data demonstrating increased
Smad3 phosphorylation in cells overexpressing YSR2 are consistent with
this possibility. Although the specific subcellular localization of the
mammalian S1P phosphatase has not been determined, yeast S1P
phosphatases have been localized to the endoplasmic reticulum (13),
consistent with the possibility of interaction of this enzyme with
TGF-
receptors or components of the TGF-
signaling pathway. In
conclusion, this study demonstrates for the first time a role for the
endogenous sphingolipid mediators in regulating the TGF-
pathway.
Further work is needed to delineate the specific mechanism of this interaction.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AR 42334 (to M. T.), AR 44883 (to M. T.), GM43825 (to Y. H.), and AG16583 (to L. O.) and the RGK Foundation.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.
§ Present address: Division of Dermatology, Saiseikai Utsunomiya Hospital, 911-1 Takebayashi-machi, Utsunomiya, Tochigi 321-0974, Japan.
** To whom correspondence should be addressed: Division of Rheumatology and Immunology, Medical University of South Carolina, 96 Jonathan Lucas St., Suite 912, Charleston, SC 29425. Tel.: 843-792-7921; Fax: 843-792-7121; E-mail: trojanme@musc.edu.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M211529200
2 M. Sato and M. Trojanowska, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
TGF-, transforming growth factor-
;
S1P, sphingosine 1-phosphate;
SPHK1, sphingosine kinase;
T
RI, TGF-
type I receptor;
ERK, extracellular
signal-regulated kinase;
COL1A1, collagen
1(I);
COL1A2, collagen
2(I);
SARA, Smad anchor for receptor activation;
YSR, yeast
sphingosine resistance;
DMEM, Dulbecco's modified Eagle's medium;
GFP, green fluorescent protein;
HA, hemagglutinin;
MEK, mitogen-activated protein kinase/extracellular signal-regulated
kinase kinase.
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