From the INSERM U317, Institut Louis Bugnard, Université Paul
Sabatier, CHU Rangueil, Batiment L3, 31403, Toulouse cedex 04, France,
the Department of Medicine, University of California, San
Francisco, California 94143-0711, and ¶ Smithkline Beecham
Laboratoires Pharmaceutiques, 35762 Saint-Grégoire, France
Received for publication, November 7, 2000, and in revised form, December 28, 2000
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ABSTRACT |
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EDG-2, EDG-4,
EDG-7, and PSP24 genes encode distinct
lysophosphatidic acid (LPA) receptors. The aim of the present study was to determine which receptor subtype is involved in the biological responses generated by LPA in preadipocytes. Growing 3T3F442A preadipocytes express EDG-2 and EDG-4
mRNAs, with no expression of EDG-7 or PSP24
mRNAs. Quantitative reverse transcriptase-polymerase chain
reaction revealed that EDG-2 transcripts were
10-fold more abundant than that of EDG-4. To determine the
involvement of the EDG-2 receptor in the responses of
growing preadipocytes to LPA, stable transfection of antisense
EDG-2 cDNA was performed in growing 3T3F442A
preadipocytes. This procedure, led to a significant and specific
reduction in EDG-2 mRNA and protein. This was
associated with a significant alteration in the effect of LPA on both
cell proliferation and cell spreading. Finally, the differentiation of
growing preadipocytes into quiescent adipocytes led to a strong reduction in the level of EDG-2 transcripts. Results
demonstrate the significant contribution of the EDG-2 receptor in the
biological responses generated by LPA in 3T3F442A preadipocytes.
Lysophosphatidic acid
(LPA1:
1-acyl-2-hydroxy-sn-glycero-3-phosphate) is a bioactive phospholipid
present in serum and other biological fluids (1, 2). LPA controls a
wide variety of cellular responses (mitogenesis, cytoskeletal
rearrangements, cell adhesion, ion transport, apoptosis) through the
activation of specific G-protein-coupled receptors (3, 4). A first potential LPA gene receptor, called vzg-1 (5), was cloned in mouse and found homologous to the endothelial differentiation gene-1
(EDG-1) (6), a high affinity receptor for another bioactive phospholipid: sphingosine 1-phosphate (7). A human gene exhibiting 97%
homology with vzg-1 was then identified and called
EDG-2 (8). Two other human genes, called EDG-4
and EDG-7, have also been cloned and proposed to be LPA
receptors (9, 10). At the beginning of the present work the cDNA
sequences of EDG-4 and EDG-7 mouse orthologues
were not yet available. Since then, the EDG-4 mouse orthologue was cloned and sequenced (11). A fourth gene receptor, called PSP24, was cloned in Xenopus (12) and
mouse (13) and initially proposed to be a LPA-responsive receptor.
However, the PSP24 receptor gene exhibits poor homology with
the EDG receptor gene family and is actually related to the
platelet-activating factor receptor gene.
Most evidence that EDG-2, EDG-4, EDG-7, and PSP24 are LPA receptors is
deduced from their ability to increase or restore LPA activity
following their overexpression in cells (5, 9, 10, 12). However, the
relative contribution of endogenously expressed LPA receptors in the
biological activities of LPA remains poorly defined. Because
pharmacological tools to study LPA receptors are very limited (no
available antagonists, difficulties in performing receptor
binding studies), one way to address the question is to alter LPA
receptor expression by using antisense strategies or gene invalidation methods.
We recently observed that conditioned medium prepared from
adipocytes exposed to an The trophic action of LPA in growing 3T3F442A preadipocytes can be
specifically desensitized by chronic exposure to a high concentration
of LPA (14). Exposure of growing 3T3F442A preadipocytes to LPA leads to
rapid and pertussis toxin-sensitive activation of the mitogen-activated
kinases ERK1 and ERK2 (15). Whereas those observations suggest the
involvement of a G-protein-coupled receptor(s) in the action of LPA in
3T3F442A preadipocytes, the identity of the discrete LPA receptor(s)
responsible for these biological effects remains to be determined.
In the present work, we studied the expression of EDG-2,
EDG-4, EDG-7, and PSP24 receptor genes
and attempted to determine their relative contribution in the
biological responses of 3T3F442A preadipocytes to LPA (proliferation
and spreading). Based upon quantification of gene expression, and
antisense cDNA transfection, the EDG-2 receptor was found to be
predominantly involved in LPA-dependent control of 3T3F442A
preadipocyte proliferation and spreading.
Cells
3T3F442A preadipocytes were grown in 10% donor calf
serum-supplemented DMEM as reported previously (16). The medium was changed every 2 days. Conversion of 3T3F442A preadipocytes into adipocytes was obtained by cultivating confluent cells in DMEM supplemented with 10% fetal calf serum plus 50 nM insulin
as described previously (14).
Nonquantitative RT-PCR Analysis
Total RNAs were extracted using RNeasy mini kit (Qiagen). One
microgram of total RNA was treated with 1 unit of RNase-free DNase I
(Life Technologies, Inc.) for 15 min at room temperature followed by
further inactivation with 1 µl of EDTA (25 mM) for 10 min
at 65 °C. Then, RNA was reverse-transcribed for 60 min at 37 °C
using SuperScript II (Life Technologies, Inc.) RNase H Oligonucleotide Primers for Nonquantitative RT-PCR
Detection of Sense mRNAs--
EDG-2 primers were
designed from mouse EDG-2 cDNA (vzg-1)
(5): sense, 5'-ATCTTTGGCTATGTTCGCCA-3' and antisense,
5'-TTGCTGTGAACTCCAGCCA-3'. Those primers are 100% identical between
mouse and human (8).
EDG-4 primers were designed from the 570-bp fragment of
mouse EDG-4 cDNA cloned in the present study (see
below): sense, 5'-TGGCCTACCTCTTCCTCATGTTCCA-3' and antisense,
5'-GGGTCCAGCACACCACAAATGCC-3'. Those primers are specific to mouse.
EDG-7 primers were designed from a GenBankTM
mouse expressed sequence tag (Clone ID 2192692, GenBankTM
accession number AW107032), which exhibits 82% identity on nucleotide level and that we assumed to correspond to mouse
EDG-7. This was confirmed later on after the identification
of the mouse EDG-7 (GenBankTM accession
number NM_012152): sense, 5'-AGTGTCACTATGACAAGC-3' and
antisense, 5'-GAGATGTTGCAGAGGC-3'. These primers are 100% identical between mouse and human.
PSP24 primers were designed such as these are homologous to
Xenopus (12), mouse (13), and human
(GenBankTM accession number HSU92642): sense,
5'-GGCCATCCTGCTCATCATTAGCG-3' and antisense,
5'-GGTGGTGAAGGCCTTGGTTTTGAA-3'.
EDG-1 primers were designed from the mouse EDG-1
gene (17): sense, 5'-GTCCGGCATTACAACTACAC-3' and antisense,
5'-TATAGTGCTTGTGGTAGAGC-3'. Those primers are 100% identical between
mouse and human (6).
Detection of Antisense mRNAs--
Detection of
EDG-2 antisense mRNAs was performed with an antisense
primer designed from the sequence of stabilization of mRNA in
pcDNA3.1 vector (5'-CAACAGATGGCTGGCAACTA-3') and a specific sense
primer designed from human EDG-2
(5'-CTGTGAAATTACAGGGATGGA-3').
Quantitative RT-PCR Analysis
cDNA was synthesized from 2 µg of total RNA in 20 µl
using random hexamers and murine Moloney leukemia virus reverse
transcriptase (Life Technologies, Inc.). A minus RT reaction was
performed in parallel to ensure the absence of genomic DNA
contamination. Design of primers was done using the Primer Express
software (Applied Biosystems). Real-time quantitative RT-PCR analyses
were performed starting with 50 ng of reverse-transcribed total RNA
with 200 nM concentration of both sense and
antisense primers in a final volume of 25 µl using the sybr green PCR
core reagents in a ABI PRISM 7700 Sequence Detection System
instrument (Applied Biosystems). Standard curves were determined after
amplification of 5 × 102 to 5 × 106
copies of purified amplicons generated from 3T3-F442A cDNA by non
quantitative RT-PCR. Quantification of EDG-2 and
EDG-4 mRNA steady state copy numbers were performed
using internal primers located within the amplicons' sequence.
Internal sense and antisense primers and size of products,
respectively, were for EDG-2 and EDG-4:
5'-CTGTGGTCATTGTGCTTGGTG-3', 5'-CATTAGGGTTCTCGTTGCGC-3', and 231 bp and
5'-GGCTGCACTGGGTCTGGG-3', 5'-GCTGACGTGCTCCGCCAT-3', and 214 bp.
Northern Blot Analysis
32P-Labeled probes were obtained by nick-translation
of cDNA fragments purified from the coding region of mouse
EDG-2 (1.1 kbp), mouse EDG-4 (0.57 kbp), mouse
EDG-1 (1.3 kbp), and mouse aP2 (0.6 kbp) genes.
Twenty µg of total RNAs were separated by electrophoresis in 1%
agarose gel containing 2.2 M formaldehyde, transferred onto a nylon membrane (Schleicher and Schuell, Dassell, Germany), and UV
cross-linked. The blot was incubated overnight at 68 °C in hybridization buffer containing 0.5 M
Na2HPO4-12H2O, 1 mM
EDTA, 7% SDS, 1% bovine serum albumin, 32P-labeled
cDNA probes, pH 7. The blot was finally washed at a final
stringency of 0.5× SSC, 0,1% SDS and autoradiographed.
Cloning of a Mouse EDG-4 cDNA Fragment
Mouse cDNAs were synthesized from total RNAs isolated from
NIH3T3 cells with random primers and reverse transcriptase. PCR was
done with degenerate primers 5'-CTiGCCiATCGCCGTiGAGCGiCA-3' and
5'-ACiACCTGiCCiGGiGTCCAGCA-3' corresponding to the third and the sixth
transmembrane domains of the human EDG-4 receptor, respectively (9).
The PCR conditions were 35 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 2 min. A product of ~570 bp was obtained and
cloned into pCR2.1-TOPO vector (Invitrogen). The sequence of this mouse
cDNA fragment was 85 and 93% identical to human EDG-4
at the nucleotide and amino acid levels, respectively. Therefore, this
cDNA fragment was assumed to correspond to the mouse
EDG-4 gene. During the time of the present study, a
full-length cDNA encoding mouse EDG-4 cDNA was
cloned (11) (GenBankTM accession number AF218844). Our
sequence was 100% homologous with this cDNA.
Stable Transfection with Antisense Vector
The coding region of human EDG-2 cDNAs (8) was
subcloned in the antisense direction in pcDNA3.1 vector
(Invitrogen). Construct was verified by restriction mapping and
sequencing. Antisense EDG-2 cDNA vector or empty
pcDNA3.1 vectors were transfected in exponentially growing 3T3F442A
preadipocytes by calcium phosphate precipitation followed by G418
(neomycin) selection as described previously (16). Gene expression and
functional analysis were performed on individual G418-resistant cell clones.
Western Blot Analysis
Preadipocyte proteins were solubilized in radioimmune
precipitation buffer, and 50 µg of protein were separated on 11%
SDS-polyacrylamide gel electrophoresis and transferred on
nitrocellulose as described previously (18). The blot was preincubated
for 2 h at room temperature in TBST buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.2% Tween 20)
containing 5% dry milk (TBST-DM) and then overnight at 4 °C in
TBST-DM containing 1/5000 anti-Vzg-1 receptor antibody (5). After extensive washing with TBST-DM, the blot was incubated with peroxidase conjugate secondary anti-rabbit antibody 1/5000e
(Sigma) for 1 h and washed again. Immunostained proteins were visualized using the enhanced chemiluminescence detection system (ECL,
Amersham Pharmacia Biotech).
Cell Proliferation and Spreading
Cell proliferation was determined as described previously (14).
After 48-h culture in 10% donor calf serum-supplemented DMEM, cells
were serum-deprived and exposed for an additional 48 h to various
growth factors such as fetal calf serum, 1-oleoyl-LPA. Cell number was
determined using Coulter counter. In some experiments LPA present in
fetal calf serum was suppressed by overnight treatment at 37 °C with
0.1 unit/ml phospholipase B (EC 3.1.1.5; Sigma).
Cell spreading was used as an index of actin cytoskeleton
reorganization and was quantified as described previously (19). Briefly, preconfluent cells were washed with phosphate-buffered saline
and placed in serum-free DMEM for 30-60 min to induce cell retraction
characterized by a reduced cell area. Cell spreading was measured by
the increase in the cell area generated after 20-min exposure to
1-oleoyl-LPA or sphingosine 1-phosphate. Cell area was measured under a
microscope connected to a video camera and image analysis program (Visiolab).
Expression of EDG Receptor mRNA in Growing 3T3F442A
Preadipocytes--
The presence of EDG-2, EDG-4,
EDG-7, and PSP24 mRNAs in total RNA prepared
from 3T3F442A preadipocytes was first tested by nonquantitative RT-PCR.
Total RNA prepared from different tissues was used in parallel as
positive control: brain (for EDG-2 and EDG-1 (5,
8, 17)), spleen (for EDG-4 (9)), heart (for EDG-7
(10)). As shown in Fig. 1,
EDG-2 and EDG-4 mRNAs, but not
EDG-7 nor PSP24 mRNAs, were detected in
3T3F442A preadipocytes. In parallel, transcripts for the sphingosine
1-phosphate receptor EDG-1 were also detected in 3T3F442A
preadipocytes.
The relative proportion of EDG-2 versus
EDG-4 mRNAs was quantified using real time RT-PCR. As
shown in Table I, EDG-2
mRNAs were found to be 10-fold more abundant than EDG-4
mRNAs (Table I). By using Northern blot analysis on total RNA,
EDG-2 (as well as EDG-1) mRNAs were easily
detectable after 24-h autoradiography (Fig.
2), whereas EDG-4 mRNAs
remained undetectable after 7 days exposure (not shown). Results showed
that although EDG-2 and EDG-4 transcripts could
be detected in growing 3T3F442A preadipocytes, EDG-2
transcripts were predominantly expressed.
Stable Transfection of Antisense EDG-2 cDNA in Growing 3T3F442A
Preadipocytes--
To determine the contribution of EDG-2 receptor in
the bioactivity of LPA, an EDG-2 antisense cDNA was
stably transfected into 3T3F442A preadipocytes. Thirteen G418-resistant
cell clones were isolated, and the presence of antisense mRNAs was
determined by RT-PCR. For that, specific primers designed for antisense
EDG-2 mRNA detection (see "Material and Methods")
were used. Six cell clones were found to express antisense
EDG-2 mRNA (Fig.
3A). Based upon Northern blot
analysis, the six clones exhibited a lower expression of endogenous
EDG-2 mRNAs as compared with 3T3F442A preadipocytes
transfected with the empty vector (Fig. 3B). Clone 7 and
clone 24 were those expressing the lowest amount of endogenous EDG-2 mRNA (Fig. 3B). It was then tested
whether down-regulation of endogenous EDG-2 mRNAs
observed in clone 7 and clone 24 was accompanied by a reduction in
EDG-2 protein level. Thus, Western blot analysis was performed with a
polyclonal antibody raised against mouse-EDG-2 receptor (Vzg-1)
(5). As described previously (5), the receptor was detected as a
protein with a molecular mass between 31 and 45 kDa (Fig.
3C). In clone 7 and clone 24, the amount of this protein was
lower when compared with 3T3F442A preadipocytes transfected with the
empty vector (Fig. 3C). No detectable reduction in the
amount of EDG-2 receptor protein was observed in clones 9, 12, and 22 (not shown). Results showed that stable transfection with
EDG-2 antisense cDNA allowed us to isolate 3T3F442A preadipocyte clones with decreased expression of endogenous EDG-2 receptor.
In parallel, we tested whether stable expression of EDG-2
antisense cDNA would affect EDG-4 receptor expression.
EDG-4 transcripts were thus quantified in the six clones
depicted in Fig. 3B. Because EDG-4 mRNAs
could not be detected by Northern blot, real time RT-PCR was used for
their quantification. When compared with 3T3F442A preadipocytes
transfected with the empty vector, the EDG-4 mRNA level
was found to be increased in clone 7, clone 19, and clone 22 and
significantly decreased in clone 12 (Table
II). Therefore, down-regulation of
EDG-2 transcripts was not systematically accompanied by a
compensatory up-regulation of EDG-4 transcripts.
Influence of EDG-2 Antisense cDNA Transfection on
LPA-dependent Proliferation--
We previously
demonstrated that 1-oleoyl-LPA, the most active LPA species, increases
proliferation in growing 3T3F442A preadipocytes (14). In 3T3F442A
preadipocytes transfected with empty vector, 1 µM
1-oleoyl-LPA by itself induced a significant increase (150% of the
control) in cell number (Fig. 4). Among
the six G418-resistant cell clones used in Fig. 3B,
only clone 7 and clone 24 exhibited a significant reduction in the
proliferative response induced by 1 µM 1-oleoyl-LPA (Fig.
4). Clone 7 and clone 24 were those harboring detectable alteration of
EDG-2 receptor protein expression (see Fig. 3C). It was also
noticeable that clone 7 exhibited an alteration of preadipocyte
responsiveness to LPA despite the existence of the high level of
expression of EDG-4 transcripts (Table II). Clone 12, which
exhibited no alteration of EDG-2 transcripts but a
significant reduction of EDG-4 transcripts (Table II),
revealed no significant alteration in the proliferative response to
LPA. Results showed that reduction of EDG-2 receptor expression was accompanied by an alteration in the proliferative response of 3T3F442A
preadipocytes to LPA. In parallel, a poor contribution of EDG-4
receptor in this response was suggested.
LPA is abundant (0.5-2.5 µM) in serum (20) and
contributes to its biological activity (21-24). In 3T3F442A
preadipocytes transfected with the empty vector, 10% serum led to a
large increase in cell number, which was significantly reduced (about
30%) by pretreatment of the serum with phospholipase B (Fig.
5). Phospholipase B is a
lysophospholipase previously shown to hydrolyze LPA and suppress its
bioactivity (14, 21, 25). Therefore, LPA significantly contributed to
the proliferative response of 3T3F442A preadipocytes to serum. In clone
7, the response to serum was significantly lower as compared with
3T3F442A preadipocytes transfected with the empty vector. In addition,
it was not significantly modified by phospholipase B treatment (Fig.
5). Similar results were obtained with clone 24 (not shown). Results
showed that clone 7 exhibited a strong reduction in its proliferative
response to the LPA present in serum. Finally, it was noticeable that
the phospholipase B-insensitive proliferative response to serum was not
significantly different between clone 7 and empty vector transfected
cells (Fig. 5). This showed that clone 7 exhibited no alteration in the
proliferative response to phospholipase B-insensitive growth factors.
Influence of EDG-2 Antisense cDNA Transfection on
LPA-dependent Spreading--
In several cell types,
including 3T3F442A preadipocytes (14, 25), 1-oleoyl-LPA (LPA) and
sphingosine 1-phosphate (S1P) (another bioactive phospholipid acting
via a distinct receptor than LPA) induce a rapid and powerful
reorganization of actin cytoskeleton, leading to a rapid spreading of
the cells previously retracted by serum deprivation (Cont in
Fig. 6C). In 3T3F442A preadipocytes transfected with empty vector (pcDNA3.1 in Fig. 6C), LPA (left panel in Fig. 6C) and
S1P (right panel in Fig. 6C) induced a
dose-dependent increase in cell spreading. This effect was
quantified by measurement of cell surface (Fig. 6, A and
B). The detectable spreading effect was observed with a 10 nM concentration of both LPA and S1P (Fig. 6,
A-C). In clone 7, the dose-response curve generated by LPA
was significantly shifted to the right, with a detectable spreading
effect observed only at 100 nM (Fig. 6, A and
C, left panel). On the contrary, the
dose-response curve generated by S1P was not significantly different
between 3T3F442A preadipocytes transfected with empty vector and clone
7 (Fig. 6, B and C, right panel). A
weak but not significant reduction in maximal response of sphingosine
1-phosphate was observed in clone 7. Results showed that clone 7 exhibited a significant and specific reduction in its spreading
response to LPA.
Expression of EDG-2 mRNAs during the Conversion of Growing
3T3F442A Preadipocytes into Growth-arrested Adipocytes--
When
cultured in fetal calf serum and insulin (see "Material and
Methods"), confluent 3T3F442A preadipocytes can be converted into
growth-arrested adipocytes (26). Conversion into adipocytes is also
characterized by a increased expression of mRNAs encoding adipocyte-specific proteins. Among them is the adipocyte-lipid-binding protein encoded by the aP2 gene (27). The influence of
adipose conversion of 3T3F442A preadipocytes was tested on the
expression of EDG-2 mRNAs. By using Northern blot
analysis, it was observed that adipocyte conversion (characterized by a
rapid increase in the aP2 mRNA level) was accompanied by
a coordinate and strong reduction in the EDG-2 mRNA
level (Fig. 7). Results suggested that
the EDG-2 receptor likely played a more important role in growing
preadipocytes than in growth-arrested adipocytes.
The results of the present study show evidence of a
predominant contribution of the EDG-2 receptor in the responses of
3T3F442A preadipocytes to LPA. Among the four potential LPA receptor
genes (EDG-2, EDG-4, EDG-7, and
PSP24), only EDG-2 and EDG-4
transcripts were found in 3T3F442A preadipocytes. Quantitative analysis
of transcript abundance revealed predominance of EDG-2 gene
receptor over EDG-4 gene receptor. This predominance was
also found in another preadipose cell line:
3T3L1.2 Therefore the EDG-2
receptor is likely primarily involved in the action of LPA in
preadipocytes. This assessment is supported by experiments showing that
antisense-directed reduction in EDG-2 receptor expression significantly
altered the responses of 3T3F442A preadipocytes to LPA. It was indeed
possible to isolate 3T3F442A-derived cell clones exhibiting significant
reduction of endogenous EDG-2 mRNA and protein level
associated with significant reduction of the cellular responses to LPA:
proliferation and cytoskeleton reorganization (Figs. 4-6). This
reduction was specific to LPA, since, in parallel, responses to
sphingosine 1-phosphate (Fig. 6) or to other phospholipase
B-insensitive growth factors (Fig. 5) were not significantly altered.
Nevertheless, this antisense strategy did not completely block the
action of LPA. This very likely resulted from a partial blockade of
EDG-2 receptor expression following antisense cDNA transfection,
since a substantial decrease, but not total disappearance, of EDG-2
expression is elicited by EDG-2 antisense stable expression. In addition, one cannot completely exclude the possible contribution of
another receptor in the residual responses generated by LPA. Besides
EDG-2 receptors, 3T3F442A preadipocytes also express EDG-4 receptor.
However, the data of Table II reveal that variations of
EDG-4 transcript expression in EDG-2
antisense-expressing clones cannot be correlated with modifications of
the proliferative response to LPA. Although we are aware that these
data should be confirmed at the protein level, they strongly suggest
the poor contribution of the EDG-4 receptor in the responses of
3T3F442A preadipocytes to LPA.
Finally, EDG-2 transcripts were predominantly expressed in
growing preadipocytes and were strongly reduced in growth-arrested adipocytes. The precise mechanisms involved in down-regulation remain unclear and are currently under investigation. Whatsoever, this
observation strongly supports the role of EDG-2 receptors in the
proliferative response to LPA in growing preadipocytes.
Although several studies have shown that overexpression of
EDG-2 cDNA restores or increases LPA sensitivity in
mammalian cells (5, 8), the specific contribution of the endogenously
expressed EDG-2 receptor remained poorly documented. Goetzl et
al. (28) have shown that the antiapoptotic response of a human
T-lymphocyte cell line to LPA could significantly be reduced by
transfection with EDG-2 plus EDG-4 antisense
cDNA. The specific contribution of the EDG-2 receptor remained to
be determined. The present study brings evidence for a specific
contribution of the EDG-2 receptor endogenously expressed in preadipocytes.
Previous work from our laboratory (14) revealed that LPA can be
produced by adipocytes and plays an important role in
paracrine/autocrine control of proximal preadipocytes. Because of the
involvement of the EDG-2 receptor in the control of the proliferation
of preadipocytes, this receptor appears to be an interesting target to
control preadipocyte proliferation, one of the key events of adipose
tissue development.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2-adrenergic stimulation
increased proliferation and spreading (reflecting a reorganization of
actin cytoskeleton) on an
2-adrenergic-insensitive
murine preadipose cell line: 3T3F442A. Analysis based on the use of a
lysophospholipid-specific phospholipase (phospholipase B) and
32P-phospholipid labeling, revealed the involvement of LPA
in the trophic activities of adipocyte conditioned medium (14).
Because of the intimate coexistence of adipocytes and preadipocytes
within adipose tissue, LPA released by adipocytes could play an
important role in paracrine/autocrine control of preadipocyte growth, a key event involved in adipose tissue development. Therefore, a better
understanding of the cellular mechanisms of LPA action in preadipocytes
could help to develop pharmacological and/or genetic strategies to
control adipogenesis.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RT
and subjected to amplification. A minus RT reaction was performed in
parallel to ensure the absence of genomic DNA contamination. PCR was
carried out in a final volume of 50 µl containing 3 µl of cDNA,
1 µl of dNTP (10 mM), 5 µl of 10× PCR buffer (10 mM Tris-HCl, pH 9, 50 mM KCl and 0.1% Triton
X-100), 3 µl of MgCl2 (25 mM), 1.5 µl of
sense- and antisense-specific oligonucleotide primers (10 µM), and 1.25 units of Taq DNA polymerase
(Promega). Conditions for the PCR reaction were: initial denaturation
step at 94 °C for 2 min, followed by 35 cycles consisting in 1 min
at 94 °C, 1 min at 54 °C (EDG-2), 57 °C
(EDG-4), 49 °C (EDG-7), 57 °C
(PSP24), or 58 °C (EDG-1), 72 °C for
90 s. After a final extension at 72 °C for 6 min, PCR products
were separated on 1.5% agarose gel, and amplification products were
visualized with ethidium bromide. In some experiments we analyzed the
influence of the number of PCR cycles on the intensity of the
amplification products.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
RT-PCR detection of EDG-2,
EDG-4, and EDG-1 mRNA in 3T3F442A
preadipocytes. RT-PCR was performed from 1 µg of DNase I-treated
total RNA (see "Material and Methods") extracted from growing
3T3F442A preadipocytes (3T3), mouse brain
(brain), mouse spleen (spleen), human heart
(heart). Thirty-five PCR cycles were performed from the same
RT with specific primers for EDG-2, EDG-4,
EDG-7, PSP24, and EDG-1 (see
"Material and Methods"). Representative result of at least three
separate experiments.
Expression of EDG-2 and EDG-4 transcripts in growing 3T3F442A
preadipocytes
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Fig. 2.
Northern blot detection of EDG-2
and EDG-1 mRNA in 3T3F442A
preadipocytes. Twenty µg of total RNA extracted from
growing 3T3F442A preadipocytes or mouse brain were analyzed by Northern
blot using specific 32P-labeled probes directed against
EDG-2 (A) and EDG-1 mRNAs
(B). An 18 S ribosomal RNA probe was used to ensure equal
well loading. Data presented are representative of at least three
separate experiments.
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Fig. 3.
Expression of EDG-2
antisense and sense mRNAs in G418-resistant cell clones
transfected with EDG-2 antisense cDNA.
A, the presence of antisense EDG-2 mRNAs was
examined by RT-PCR analysis (see "Material and Methods") in
G418-resistant cell clones (clones 7 to 31) transfected with antisense
EDG-2 cDNA and in empty pcDNA3.1 transfected cells.
B, EDG-2 mRNA level in G418-resistant cell
clones analyzed by Northern blot as described in the legend to Fig. 2.
C, EDG-2 receptor protein level analyzed by Western blot
(see "Material and Methods") in clone 7, clone 24, and in empty
pcDNA3.1 transfected cells: representative of two separate
experiments.
Expression of EDG-4 transcripts in 3T3F442A preadipocytes transfected
with EDG-2 cDNA antisense
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Fig. 4.
Influence of LPA on the proliferation of
G418-resistant cell clones transfected with EDG-2
antisense cDNA. Each cell clone was seeded and grown in
10% donor calf serum-supplemented DMEM. After 48 h, serum was
removed, and each cell clone was grown for an additional 48 h in
the presence or absence of 1 µM 1-oleoyl-LPA. Cell number
obtained in each clone was determined as described under "Material
and Methods" and compared with that obtained with empty pcDNA3.1
transfected cells. Each column represents the mean ± S.E. of
three to five independent experiments, depending on cell clone.
Statistical analysis was performed using the Student's t
test: *, p < 0.05 when comparing LPA activity in each
clone to that measured in empty pcDNA3.1 transfected cells.
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Fig. 5.
Influence of antisense EDG-2
cDNA transfection on the proliferative response to
serum. Clone 7 and empty pcDNA3.1 transfected 3T3F442A
preadipocytes were seeded and grown in 10% donor calf
serum-supplemented DMEM. After 48 h, the medium was changed with
fresh 10% fetal calf serum-supplemented DMEM pretreated (+) or not
( ) with 0.1 unit/ml phospholipase B overnight. Cell number
was determined after 48 h as described under "Material and
Methods." Each column represents the mean ± S.E. of five
independent experiments. Statistical analysis was performed using the
Student's t test: p < 0.05 when comparing
the effect of serum with that of phospholipase B-treated serum (*) and
p < 0.05 when comparing pcDNA3.1 with clone 7 in
the absence of phospholipase B (#). NS, nonstatistically
different.
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Fig. 6.
Influence of EDG-2 antisense
cDNA expression on the cell spreading response to LPA. Growing
clone 7 (black boxes) or empty pcDNA3.1 transfected
cells (white boxes) were retracted by serum deprivation as
described under "Material and Methods" and exposed to increased
concentrations of 1-oleoyl-LPA (A) or sphingosine
1-phosphate (B). After 15 min the cell surface was measured
as the intensity of cell spreading. Each value represents the mean ± S.E. of three independent experiments. Statistical analysis was
performed using the Student's t test: *, p < 0.05 when comparing clone 7 (black boxes) and empty
pcDNA3.1 transfected cells (white boxes). C,
photo of one representative experiment of cell spreading.
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Fig. 7.
Expression of EDG-2 and
aP2 mRNAs during conversion of 3T3F442A
preadipocytes into adipocytes. 3T3F442A preadipocytes were
grown in donor calf serum (SVD)-supplemented DMEM until
confluence. At confluence, the medium was replaced by fetal calf
serum-supplemented DMEM plus insulin (SVF+insulin) to induce
adipose conversion (see "Material and Methods"). Total RNAs were
extracted at different time during the course of preadipocytes
conversion into adipocytes, and mRNAs were detected by Northern
blot. Data are representative of at least three separate
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Jerold Chun for providing Vzg-1 antibody, Dr. Gabor Tigyi for providing Xenopus PSP24 vector and mouse PSP24 sequence, and Isabelle Lefrère for expert technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the "Institut National de la Santé et de la Recherche Médicale" (APEX 4 × 405D), the "Association pour la Recherche sur le Cancer" (5381), the "Laboratoires Clarins" and the "Institut de Recherche Servier."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: Tularik Inc., Two Corporate Dr., South San Francisco, CA. 94080. E-mail: san@tularik.com.
To whom correspondence should be addressed. Tel.:
33-0562172956; Fax: 33-0561331721; E-mail:
saulnier@rangueil.inserm.fr.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M010111200
2 S. Krief, personal data.
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ABBREVIATIONS |
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The abbreviations used are: LPA, lysophosphatidic acid; DMEM, Dulbecco's modified Eagle's medium; RT-PCR, reverse transcriptase-polymerase chain reaction; bp, base pair(s); kbp, kilobase pair(s); S1P, sphingosine 1-phosphate.
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REFERENCES |
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