|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1701-1710
Interleukin-9-Induced Expression of M-Ras/R-Ras3 Oncogene in T-Helper
Clones
By
Jamila Louahed,
Luigi Grasso,
Charles De Smet,
Emiel Van Roost,
Claude Wildmann,
Nicholas C. Nicolaides,
Roy C. Levitt, and
Jean-Christophe Renauld
From the Ludwig Institute for Cancer Research, Brussels Branch, and
the Experimental Medicine Unit, Université Catholique de Louvain,
Brussels, Belgium; and Magainin Institute of Molecular Medicine,
Plymouth Meeting, PA.
 |
ABSTRACT |
In an attempt to gain insight into the molecular mechanisms involved
in interleukin-9 (IL-9) activities, representational difference
analysis (RDA) was used to identify messages that are induced by IL-9
in a murine T-helper-cell clone. One of the isolated genes encodes for
the newly described M-Ras or R-Ras3, which is part of the Ras gene
superfamily. M-Ras expression was found to be induced by IL-9 but not
IL-2 or IL-4 in various murine T-helper-cell clones, and this
induction seems to be dependent on the JAK/STAT pathway. Contrasting
with the potent upregulation of M-Ras expression, M-Ras was not
activated by IL-9 at the level of guanosine triphosphate/guanosine diphosphate (GTP/GDP) binding. However, IL-3 increased GTP
binding to M-Ras, suggesting that M-Ras induction might represent a new mechanism of cooperativity between cytokines such as IL-3 and IL-9.
Constitutively activated M-Ras mutants induced activation of Elk
transcription factor by triggering the MAP kinase pathway and allowed
for IL-3-independent proliferation of BaF3 cells. Taken together,
these results show that cytokines such as IL-9 can regulate the
expression of a member of the RAS family possibly involved in
growth-factor signal transduction.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
INTERLEUKIN-9 (IL-9) is a TH2 cytokine
that was discovered based on its growth-promoting effect on T-helper
cells.1 Subsequently, several other activities were
attributed to this protein, including IgE production by B cells,
differentiation of hematopoietic and neuronal progenitor cells, and
proliferation as well as differentiation of mast cells.2,3
In addition, some observations suggest the involvement of IL-9 in both
human and murine tumorigenesis. IL-9 overexpression indeed results in a
high susceptibility to the development of T-cell lymphomas in vivo,4 and constitutive IL-9 expression has been shown in
many human Hodgkin lymphomas.5 More recently, the
involvement of IL-9 has been suggested in asthma,6-9 in
line with the effect of this cytokine on IgE
production10,11 and mast cell
differentiation.12
To further characterize the biological activity of IL-9, we established
a panel of T-helper-cell clones that can proliferate in response to
either IL-2, IL-4, or IL-9, and thus provide us with a simple model to
compare functional characteristics of these cytokines.13 In
this report, using 1 of these T-cell clones, we employed a subtractive
hybridization approach to identify genes whose expression is induced by
IL-9 and may be involved in the IL-9 pathway to regulate proliferation
and/or apoptosis in T-helper cells. Using this strategy, we found that
the expression of the guanosine triphosphate (GTP)-binding
protein M-Ras can be selectively induced by IL-9 but not by IL-2 or
IL-4. Biochemical analysis of constitutively expressed M-Ras in
lymphoid cells failed to detect its activation by IL-9 in contrast to
IL-3, suggesting that this molecule is not involved in IL-9 signal
transduction in effector cells. However, these data suggest a mechanism
by which M-Ras activity can be regulated at the mRNA level in addition to the posttranslational level of GTP binding and GTPase activity.
| |
MATERIALS AND METHODS |
Cell cultures and cytokines.
T-helper-cell clones TS2 and TS313 were grown in
Dulbecco's modified Eagle's medium (DMEM) medium
supplemented with 10% fetal calf serum (FCS), 50 µmol/L
2-mercaptoethanol, 0.55 mmol/L L-arginine, 0.24 mmol/L L-asparagine,
and 1.25 mmol/L L-glutamine. These factor-dependent cell lines were
able to grow in the presence of either IL-2, IL-4, or IL-9 without
antigen and feeder cells.1 TS2-h9R cells were obtained by
electroporation of TS2 cells with the pEF-BOS.puro vector containing
the wild-type human IL-9 receptor cDNA and selection in puromycin (4 µg/mL; Sigma, St Louis, MO) and human IL-9 (500 U/mL).
TS2-h9Rphe116 cells were derived using the same procedure with a
mutated human IL-9 receptor cDNA, where Tyr116 of the cytoplasmic tail
is changed to a Phe residue. As described previously, this mutation
abolished STAT (signal transducer and activator of
transcription) activation in response to human IL-9.14 BaF3
cells15 were cultured in DMEM supplemented with 10% FCS
and mouse IL-3 (1 ng/mL). HEK293 cells, derived from human embryonic
kidney, were cultured in DMEM medium with 10% FCS. The following cell
lines were kindly provided as listed: T-helper clone
ST2K916 by Dr E. Schmitt (Gütenberg University,
Mainz), IL-9- or IL-3-dependent mast cell line L138.8A12
by Dr L. Hültner (GSF, München, Germany), IL-3-dependent
mast cell line MC9 by Dr C. Petit-Frère (Institut Henri Beaufour,
Les Ullys, France), macrophage cell line PU5.8 by Dr L. Franssen
(Innogenetics, Gent, Belgium), and thymic lymphoma EL4 by Dr H.R.
MacDonald (Ludwig Institute for Cancer Research, Lausanne, Switzerland).
Spleen cells were isolated from naive 12-week-old C57BL/6 mice by
aseptic removal of spleen and resuspension in DMEM supplemented with
10% heat-inactivated FCS. Cells (106/mL) were incubated
with or without 5 µg/mL of Conconavalin A (Sigma). After 24 or 48 hours, cells were harvested for RNA isolation using the Trizol reagent
(GIBCO-BRL, Ghent, Belgium) according to the
manufacturer's recommendations.
Mouse and human recombinant IL-9 (4 × 107 U/mg and 2 × 107 U/mg, respectively) were produced in our
laboratory by expression in the baculovirus system and purified as
described.17 Human recombinant IL-2 (3.5 × 106 U/mg) was given by Dr W. Fiers (State University of
Ghent, Ghent, Belgium), and mouse recombinant IL-3 (2 × 107 U/mg) was a gift of Dr J.Y. Bonnefoy (Glaxo, Geneva, Switzerland).
Subtractive hybridization.
Total RNA was prepared from TS2 cells stimulated with IL-2 (200 U/mL)
or IL-9 (200 U/mL) for 48 hours, using guanidium isothiocyanate lysis
and CsCl gradient centrifugation.18 Polyadenylated RNA was
purified from total RNA with oligo(dT) cellulose columns. Double-stranded cDNA was generated from 5 µg polyA+ RNA using an
oligo(dT) primer and the SuperScript Choice System for cDNA synthesis
according to the manufacturer's recommendations (GIBCO-BRL). Representational difference analysis was performed as described by
Hubank and Schatz19: cDNAs were digested with DpnII
(New England Biolabs, Beverly, MA), ligated to R-Bgl-12/24
adapters, and polymerase chain reaction (PCR) amplified to generate
representations. The R-Bgl oligonucleotides were removed by
DpnII digestion, and J-Bgl-12/24 adapters were ligated to the
cDNA from TS2/IL-9 cells. For the first cycle of substractive
hybridization, 0.4 µg of J-Bgl-ligated TS2/IL-9 cDNA was mixed with
40 µg TS2/IL-2 cDNA (1/100 ratio). After hybridization for 20 hours
at 67°C, the ends of the resulting hybrids were filled in, and a
PCR amplification was performed with the J-Bgl-24 oligonucleotide,
which was further removed by DpnII digestion. The second and
third cycles of subtraction were performed similarly with a ratio of
1/800 and 1/400,000, respectively.
After these 3 rounds of subtraction, final difference products were
digested with DpnII and cloned into the BamHI site of pTZ19R. Double-stranded plasmid DNA was prepared and sequenced with a
Thermo-sequenase Sequencing kit (Amersham, Arlington Heights, IL).
Sequence comparisons with the GenBank and European Molecular Biology
Laboratory (EMBL) databases were performed with the BLAST search program. Oligo(dT)-primed cDNA libraries generated from the
mouse T-cell clone TS2 stimulated with IL-9, and from human testis were
screened with the mouse M-Ras DpnII fragment and with a human
reverse transcription (RT)-PCR product, respectively.
Preparation of mRNA, Northern, and RT-PCR analysis.
Total cellular RNA was fractionated by electrophoresis in a 1.3%
agarose gel containing 2.2 mol/L formaldehyde and was transferred onto
a Hybond-C Extra nitrocellulose membrane (Amersham). cDNA inserts were
labeled using the Multiprime DNA labeling kit from Amersham.
Hybridizations and washes were performed as described.13 The M-Ras probe was a 1.2-kb cDNA containing the complete coding sequence for this protein. After autoradiography, all blots were reprobed with a chicken  -actin probe to control for even loading of
RNA. To analyze the tissue distribution of M-Ras expression, a premade
nitrocellulose multiple tissue filter covering 8 tissues was used
(Clontech, Palo Alto, CA).
RT was performed on 5 µg total RNA with an oligo(dT) primer. cDNA
corresponding to 20 ng of total RNA was amplified for 20 to 35 cycles
by PCR with specific primers as indicated in
Table 1. The post-PCR products were
analyzed in ethidium bromide-stained 1% agarose gel. Specific
amplification was confirmed after blotting (Zeta-Probe membrane;
Biorad, Hercules, CA) and hybridization of the post-PCR
results with internal radioactive probes. For -actin the internal
probe is 5'-GTCCACGACATCATGCTACTG-3', and for the mouse
M-Ras, 5'-GGATGTTCTGGACACAGCCGG-3'. Radioactive signals
were quantified by Phosphorimager (Molecular Dynamics, Sunnyvale,
CA) and the M-Ras/actin ratios were calculated. Human M-Ras was amplified from a human brain cDNA with the oligonucleotides designed from the mouse sequence and shown in Table 1.
M-Ras-specific antibody production.
A synthetic peptide beginning with an NH2-terminal cysteine residue
followed by the M-Ras sequence 187-204 (CKKKTKWRGDRATGTHKLQ), a region
that showed no homology with other members of the Ras family, was
conjugated to maleimide-activated KLH (Imject-Maleimide activated
Carrier proteins; Pierce, Rockford, IL). Two rabbits were
each immunized with 150 µg of peptide-KLH conjugate in 4 vol of
Freund complete adjuvant by multiple-site intradermal injection. At
3-week intervals, the rabbits were boosted with 100 µg of
peptide-conjugate in 4 vol of Freund incomplete adjuvant by multisite
subcutaneous injection. Fifteen days after the second boost, the
animals were test bled, and after the third boost the recovery period
between injections was increased to 4 weeks, with test bleedings 10 to 14 days post-boost. The production of peptide-specific antibodies (JAL-4) was assessed by enzyme-linked immunosorbent assay (ELISA).
Electrophoretic mobility shift assay.
Nuclear extracts were prepared as described,20 with minor
modifications. Briefly, cells (1.5 × 107) stimulated
with murine (m) IL-2 (200 U/mL), mIL-9 (200 U/mL), or
hIL-9 (500 U/mL) for 10 minutes, were washed with phosphate-buffered saline (PBS), and resuspended in 1 mL ice-cold hypotonic buffer `A'
for 15 minutes (10 mmol/L HEPES buffer, pH 7.5, containing 10 mmol/L
KCl, 1 mmol/L MgCl2, 5% glycerol, 0.5 mmol/L EDTA, 0.1 mmol/L EGTA, 0.5 mmol/L dithiothreitol, 1 mmol/L Pefabloc [Boehringer Mannheim, Mannheim, Germany], 10 mg/mL aprotinin, 1 mmol/L
Na3VO4, 5 mmol/L NaF). The cells were lysed by
adding 65 mL NP-40 10% and vortexing. Nuclei were pelleted (30 seconds
at 14,000 rpm) and extracted for 30 minutes in 100 mL hypertonic buffer
`B' (buffer `A' supplemented with HEPES [20 mmol/L], glycerol
[20%], and NaCl [420 mmol/L]). Nuclear debris were removed through
a 2-minute centrifugation. Analysis of DNA binding activity was
performed using 32P-labeled GRR oligonucleotide probes
(from Fc RI gene promoter): upper strand:
5'-ATGTATTTCCCAGAAA-3'; bottom strand:
5'-CCTTTTCTGGGAAATAC-3'.
Isolation of BaF3 transfectants.
cDNA encoding wild-type (M-Ras), activated mutants of mouse M-Ras
(M-Ras/22V, M-Ras/22K, or M-Ras/71K) were cloned into the pEF-BOS
plasmid,21 in which a puromycin resistance gene had been
inserted for selection of transfected cells.14 Mutagenesis was performed in pBluescript using appropriate oligonucleotides followed by subcloning into pEF-BOS (Stratagene, La Jolla,
CA; Chameleon Double-Stranded, site-directed Mutagenesis
Kit) and the coding sequence of each mutant was checked with a
Thermo-sequenase Sequencing kit (Amersham). Plasmid DNA was extracted
with a Nucleobond AX kit (Macherey-Nagel, Duren, Germany).
BaF3 were transfected by electroporation (1,500 µF, 74 W, and 300 V)
with 50 µg of sterile DNA. Pools of transfected cells were selected
with puromycin (3 µg/ml; Sigma). M-Ras and M-Ras/22V-transfected BaF3 cells were further transfected with pRG8 plasmid containing the
human IL-9 receptor cDNA and selected with G418 (1 mg/mL; GIBCO-BRL).
Selected cells were maintained in growth medium supplemented with 20 ng/mL human IL-9 or 1 ng/mL of murine IL-3.
GTP-, GDP-bound RAS assay.
Cells were washed 3 times in PBS, resuspended in LB (DMEM without
sodium phosphate or sodium pyruvate [GIBCO-BRL] containing 20 mmol/L
HEPES, 0.1% bovine serum albumin [BSA], and 0.3 mCi/mL of
phosphorus-32 [Amersham]), and incubated for 4 hours at 37°C. Two
to 3 × 107 cells were used for each experimental
point. Cells were stimulated for 5 minutes with the appropriate
cytokine and immediately washed with ice-cold PBS. Cells were lysed in
1 mL of buffer 1 (50 mmol/L HEPES pH 7.5, 100 mmol/L NaCl, 01% Triton
X-114 [Sigma], 5 mmol/L MgCl2, 0.1% BSA, 1X
"Complete" protease inhibitor mix [Boehringer Mannheim]) and
lysates were cleared by centrifugation. Supernatant was transferred in
a fresh tube containing 100 µL of 5 mol/L sodium chloride, incubated
for 2 minutes at 37°C, and centrifuged. The aqueous layer was
discarded and the detergent layer (50 µL) was resuspended in 1 mL of
buffer 2 (50 mmol/L Tris pH 7.5, 5 mmol/L MgCl2, 1% Triton
X-100, 0.5% deoxycholate, 0.05% sodium dodecyl sulfate [SDS], 0.5 mol/L NaCl, 1 mmol/L EGTA pH 8.0, 1 mmol/L dithiothreitol [DTT],
0.1% BSA, 1X "Complete" protease inhibitor mix) and 25 µL of
protein A+G agarose-conjugated beads were added. Lysates were
precleared for 5 minutes. The anti-M-RAS antiserum (JAL-4) was added
(1/100) and lysates were tumbled for 45 minutes at 4°C.
Subsequently, 20 µL of protein A+G agarose-conjugated beads were
added and lysates were tumbled at 4°C. Beads were washed 5 times in
washing buffer (50 mmol/L HEPES pH 7.5, 0.5 mol/L NaCl, 0.1% Triton
X-100, 0.005% SDS, 5 mmol/L MgCl2), resuspended in 12 µL
of elution buffer (2 mmol/L EDTA, 2 mmol/L DTT, 0.2% SDS, 5 mmol/L GTP
[Sigma], 5 mmol/L guanosine diphosphate [GDP; Sigma]) and incubated
at 68°C for 20 minutes. Samples were then centrifuged and 8 µL
was loaded in a PEI-cellulose TLC plate (Merck, Darmstadt, Germany) that was subsequently developed in 0.75 mol/L
potassium phosphate pH 3.4 for 1.5 hours.
Western blot.
Cells (2 × 105) were lysed in modified RIPA buffer
(Tris HCl 50 mmol/L, pH 8, NaCl 150 mmol/L, Triton X-100 1%, sodium
deoxycholate 0.25%, EGTA 1 mmol/L, NaF 1 mmol/L, leupeptin 10 mg/mL,
Pefabloc 2 mmol/L) and cell debris was removed by centrifugation.
Lysates were fractionated on SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) polyacrylamide gels (14%) and
electrophoretically transferred onto PVDF membranes
(Amersham). Membranes were blocked in 5% nonfat milk, washed, and
probed with rabbit polyclonal M-Ras antiserum (JAL-4, 1/500) and with
horseradish peroxidase-linked anti-rabbit antibody (1/5,000;
Amersham). The ECL detection kit (Amersham) was used for detection.
Signal transduction pathway assay.
Wild-type or mutated M-Ras constructs were cotransfected using a lipid
transfection method into HEK293 cells together with the
pathway-specific trans-activator vector and the reporter vector (Pathdetect trans-reporting system; Stratagene). The HEK293 cells were
seeded at a density of 5 × 105/well in 12-well
tissue-culture plates, and incubated overnight at 37°C. Each assay
was performed with 3 plasmids: (1) 25 ng of the fusion activator
plasmid (pFA vector) encoding a pathway-specific trans-activator
protein consisting of the DNA binding domain of the yeast GAL4 and the
activation domains of Elk; (2) 500 ng of the pFR-Luc reporter plasmid
in which expression of the luciferase gene is controlled by a synthetic
promoter that contains the yeast GAL4 binding site; (3) 500 ng of
either empty pEF-BOS or pEF-BOS plasmid containing wild-type or mutated
M-Ras. pFC-MEK1 plasmid (25 ng) encoding constitutively activated
kinase was used as positive control plasmid for the MAPK-pathways.
pFC-dbd (encoding the GAL4 DNA-binding domain) was used as a negative
control for the pFA vector to ensure that the observed effects were not
due to the GAL4 DNA binding domain.
Target cells were washed once with serum-free medium (OPTI-MEM; GIBCO),
and transfected with the DNA constructs in 50 µL serum-free medium
that had been mixed with 20 µg of lipofectamine reagent (GIBCO-BRL)
in 50 µL of OPTI-MEM medium. Twenty-four hours after transfection,
cells were washed twice with PBS before lysis and analysis of
luciferase activity using the "Luciferase Reporter Gene Assay"
kit according to the manufacturer's instructions (Boehringer Mannheim).
| |
RESULTS |
Isolation of an IL-9-induced gene homologous to Ras proto-oncogenes.
Although IL-9 and IL-2 have T-cell growth activity for mouse T-helper
clones, some experiments suggest that distinct activation pathways are
involved.22 Based on the hypothesis that different biological activities of these cytokines reflect their ability to
transcriptionally activate distinct target genes in T cells, we
performed a representational difference analysis of gene expression on
TS2 cells that were stimulated with either IL-2 or IL-9.
Oligo(dT)-primed cDNAs prepared from cells stimulated with both
cytokines were digested with DpnII and used to generate the
respective amplicons. After 3 rounds of subtractive hybridization, the
third difference product (DP3) was cloned and 57 clones were sequenced.
Five independent IL-9-induced genes were identified, one of them
showing a significant sequence identity with members of the Ras family.
To obtain the full-length sequence of this mRNA, we screened an
oligo-dT primed cDNA library generated from IL-9-stimulated TS2 cells
with a 200-bp DpnII restriction fragment recovered from the
subtractive hybridization. The largest clone isolated contained 1,128 nucleotides including a 624-bp open reading frame (ORF) that encodes
for a 208 amino acid residue protein with a molecular weight
(Mr) of 23,886 (GenBank accession no. AF043581).
A human homologue was identified from human brain cDNA by RT-PCR
amplification using oligonucleotides based on the mouse sequence. Several cDNA clones were subsequently isolated from a human testis cDNA
library. The largest of these cDNA clones contained a 1,081-bp insert
with an ORF that encodes a protein of 208 amino acids with a calculated
molecular mass of 23,831 daltons (GenBank accession no.
AF043938). Sequence alignment of the mouse and human genes showed an
identity of 89% at the nucleotide level and 97% at the protein level.
During the characterization of these genes, the cloning of the mouse
and human cDNAs was independently reported by 2 groups, based on
homologies with other Ras genes.23,24 When compared with
our sequence, the published mouse M-Ras cDNA sequence differs by only 3 nucleotide substitutions in the 3' untranslated region. The human
M-Ras cDNA shows 3 differences in the coding sequences and 2 of them
result in a change of the amino acid sequence (glutamic acid-166 is
replaced by a lysine and alanine-199 by a glycine in our sequence).
These discrepancies may result from the low fidelity of Taq polymerase
since the published M-Ras/R-Ras3 sequences were obtained by PCR
amplification, whereas our sequences were found in several cDNA clones
from conventional cDNA libraries. Alternatively, this could correspond
to true allelic variations of the same gene.
A comparison of the amino acid sequence of murine M-Ras with those of
murine H-Ras and R-Ras showed an amino acid identity of 49% and 46%
with p21 H-Ras and p23 R-Ras, respectively. Conserved residues include
an amino-terminal domain with a putative GTP-binding site (amino acids
20-27) corresponding to amino acids 10 to 17 in the H-Ras sequence. The
carboxy-terminus was characterized by the presence of the consensus
prenylation signal CAAX (where C is a cysteine, A is a hydrophobic
residue, and X can be any residue) known to play an important role in
the plasma membrane localization of Ras.25 In addition, a
cluster of 8 basic residues (lysine) located upstream of this motif may
mediate membrane attachment by electrostatic interaction with acidic
phospholipids on the inner leaflet membrane, as shown previously for
K-Ras.26
IL-9 inducibility of mouse M-Ras expression.
To confirm the inducibility of M-Ras by IL-9, we analyzed a series of
IL-9-responsive cell lines including T-helper clones (TS2, TS3, and
ST2K9) and mast cell lines (MC9 and L138). The murine M-Ras transcripts
(2 bands of 1.3 and 4.4 kb) were observed in the T-cell clone TS2
cultured in the presence of IL-9 whereas neither IL-2- nor
IL-4-stimulated cells had detectable amounts of M-Ras transcript
(Fig 1). Induction of M-Ras expression by IL-9, but not by IL-2, was confirmed in 2 additional T-helper-cell clones (TS3 and ST2K9). By contrast, we failed to detect M-Ras expression in the mast cell lines MC9 and L138 stimulated with IL-9,
indicating that IL-9 induced the expression of this gene in only a
subset of IL-9-responsive cells. The inducibility by IL-9 is a
specific characteristic of M-Ras since other members of the Ras family,
such as H-Ras, N-Ras, and R-Ras, were expressed at similar levels in
TS2 cells stimulated by IL-9 or IL-2 (Fig 2).
View larger version (30K):
[in this window]
[in a new window]
| Fig 1.
IL-9 induces expression of M-Ras. IL-9-responsive cell
lines were cultured for 10 days in medium containing saturating
concentrations of the indicated cytokines: 100 U/mL IL-2, 200 U/mL
IL-4, or 200 U/mL IL-9 for T-helper-cell clones (TS2, ST2K9, and TS3),
and 200 U/mL of IL-3 or 200 U/mL of IL-9 for mast cell lines (L138 and
MC9). After electrophoresis of 10 µg of total RNA and transfer on
nitrocellulose, filters were hybridized with a 32P-labeled mouse M-Ras
cDNA probe. Hybridization with a -actin probe confirmed that
comparable amounts of RNA had been loaded in each lane.
|
|
View larger version (52K):
[in this window]
[in a new window]
| Fig 2.
IL-9 does not regulate expression of other members of the
Ras family. TS2 cells were cultured for 3 days in the presence of 100 U/mL IL-2 or 200 U/mL IL-9. Total RNA was extracted and RT-PCR
amplification was performed as described in Materials and Methods,
using specific oligonucleotides listed in Table 1. Amplifications were
performed at 94°C for 30 seconds, 58°C to 62°C for 1 minute, 72°C for 1.5 minutes, with a total number of 18 cycles for
-actin or 35 cycles for M-, H-, N-, and R-Ras. Samples where the
reverse transcriptase (RT) was omitted were used as negative controls
for each condition. The post-PCR products were analyzed in ethidium
bromide-stained 1.5% agarose gel. The specificity of the PCR
amplification was checked by sequencing the PCR products.
|
|
The kinetics of IL-9-induced M-Ras induction in TS2 cells found its
expression increased at 6 hours and reached a maximal level at 24 hours
(Fig 3). We next investigated the
expression of M-Ras in different tissues and found that the highest
expression of M-Ras level could be detected in brain, although a
significant M-Ras expression was also found in kidney and skeletal
muscle. In the other tissues tested, M-Ras messenger was barely
detectable (Fig 4A). Tissue expression was
also analyzed on IL-9 transgenic mice (Tg5) that overexpress IL-9 in
all tissues (data not shown) and in its congenic background strain
(FVB). No significant difference was observed between normal and IL-9
transgenic mice (Fig 4B). Noticeably, IL-9 overexpression in these
transgenic mice does not induce normal T-cell hyperplasia, probably
because resting T cells do not respond to IL-9. By contrast, IL-9
induces intestinal mastocytosis with increased expression of other
IL-9-regulated genes such as granzymes and proteases.27
Thus, the lack of M-Ras upregulation in the gut of IL-9-transgenic
mice supports the observation made on cell lines that IL-9 induction of
M-Ras was restricted to some cell types and/or conditions, such as
activated T cells. In this respect, M-Ras induction is not limited to
IL-9-stimulated T cells, as this gene is also upregulated upon ConA
stimulation of freshly isolated spleen cells
(Fig 5).
View larger version (22K):
[in this window]
[in a new window]
| Fig 3.
Kinetics of induction of M-Ras in TS2 cell. TS2 cells
growing in the presence of IL-2 (100 U/mL) were washed and incubated
with 200 U/mL of IL-9. Two independent experiments are shown. (A)
Northern blots were prepared with 10 µg of total RNA isolated from
cells stimulated for the indicated times, in hours (h) or in days (d).
Filters were probed with a 32P-labeled mouse M-Ras cDNA.
(B) M-Ras expression was monitored by RT-PCR and normalized with
-actin expression as described in Materials and Methods.
M-Ras/-actin ratios are arbitrary units.
|
|
View larger version (39K):
[in this window]
[in a new window]
| Fig 4.
Tissue distribution of mRNA for mouse M-Ras. (A) Northern
blot of a multiple tissue [2 µg of mouse poly(A)+ RNA per lane]
(Clontech) was hybridized with a 32P-labeled mouse M-Ras
cDNA probe. The size of the mRNAs detected are indicated to the left of
the panel. (B) Northern blots containing 10 µg per lane of total RNA
from adult tissues of FVB mice or IL-9-transgenic mice (Tg5) were
hybridized with a 32P-labeled mouse M-Ras cDNA probe.
Ethidium bromide staining of the gel is shown as control for loading.
|
|
View larger version (39K):
[in this window]
[in a new window]
| Fig 5.
Induction of M-Ras expression by ConA stimulation of
freshly isolated spleen cells. Spleen cells were cultured for 24 hours
with or without ConA (5 µg/mL). Total RNA was extracted and RT-PCR
amplification was performed as described for Fig 2. Samples where the
reverse transcriptase (RT) was omitted were used as negative controls
for each condition. The post-PCR products were analyzed in ethidium
bromide-stained 1.5% agarose gel.
|
|
STAT proteins mediate IL-9 induction of M-Ras.
To determine the mechanisms by which IL-9 upregulate M-Ras, we
transfected TS2 cells with the wild-type human IL-9 receptor (h9R) or
its mutated form, that lacks the ability to activate STAT transcription
factor due to substitution of a single tyrosine residue with
phenylalanine (h9Rphe116). This residue is known to be phosphorylated
by JAK tyrosine kinases and to serve as a docking site for STAT
transcription factors.14 Both tranfectants, but not the
parental cells, were able to proliferate in response to human IL-9
(data not shown). As shown in Fig 6A, hIL-9
activated STAT transcription factors (STAT-1, -3, -5) only in cells
expressing the wild-type human IL-9R, and not in cells expressing the
phenylalanine mutant. The expression of M-Ras was induced by hIL-9 only
in cells expressing the wild-type receptor (Fig 6B). These data suggest that STAT activation is required for M-Ras upregulation.
View larger version (82K):
[in this window]
[in a new window]
| Fig 6.
IL-9-induced M-Ras expression in TS2 cells expressing
wild-type or mutant hIL-9R. (A) Electromobility shift assay using a
STAT-binding oligonucleotide. TS2 transfectants were stimulated for 10 minutes with IL-2, mIL-9, or hIL-9 (which does not bind the endogenous
murine receptor) and nuclear extracts were prepared, and the
electromobility shift assay was performed with the GRR oligonucleotide
as described in Materials and Methods. (B) Northern blot analysis of
M-Ras expression. The same cells were cultured for 48 hours in the
presence of 200 U/mL of either IL-2 or murine IL-9, or 500 U/mL of
human IL-9. Northern blots were prepared with 10 µg of total RNA and
hybridized as described in Fig 1.
|
|
IL-3 but not IL-9 induces activation of M-Ras.
Binding of cytokines such as IL-2 or IL-3 to their receptors has been
shown to cause activation of Ras-p21. To study whether IL-9 could also
trigger activation of M-Ras, BaF-3 cells constitutively expressing
wild-type M-Ras were further transfected with the human IL-9 receptor.
Cells were starved in serum-free medium before stimulation with mIL-3
or human (h) IL-9, M-Ras was immunoprecipitated, and the
GTP/GDP binding was analyzed by thin-layer chromatography. Figure 7 shows that IL-9 is unable to
activate M-Ras while IL-3 stimulation increased the amount of GTP-bound
M-Ras, thus showing that IL-9 does not use this Ras pathway to mediate
signaling in downstream effector cells. These data are concordant with
the finding that IL-9 does not elicit activation of Ras-p21 in lymphoid cells (Grasso L, Demoulin JB, Atkins JM, Louahed J, Renauld JC, Levitt
RC, Nicolaides NC: submitted for publication) and
suggests a potential synergy between IL-9 and IL-3, where IL-9 induces the expression of M-Ras, which in turn can be activated by another growth factor.
View larger version (55K):
[in this window]
[in a new window]
| Fig 7.
Activation of M-Ras by IL-3 but not by IL-9. BaF3 cells
constitutively expressing the hIL-9 receptor and wild-type M-Ras were
cytokine-starved 8 hours in serum-free medium before stimulation with
500 U/mL of either hIL-9 or 3 ng/mL of mIL-3. Thin-layer chromatography
of in vivo 32P-labeled GDP and GTP was perfomed after
immunoprecipitation of M-Ras. The result is representative of 3 independent experiments. untreat, untreated cells.
|
|
M-Ras activates the MAPKinase pathway.
To further elucidate the biological role of M-Ras in lymphoid cells, we
generated murine cDNAs carrying the oncogenic mutations on codons 22 or
71. The M-Ras Gly 22 was replaced by a valine (M-Ras/22V) or a lysine
(M-Ras/22K), and M-Ras Glu 71 was replaced by a lysine (M-Ras/71K). The
corresponding mutants of p21 H-Ras have been shown to persist in the
GTP-bound form, which is the active form of
Ras.28 Figure 8 shows a
thin-layer chromatography of in vivo 32P-labeled GDP and
GTP eluted from M-Ras immunoprecipitated from BaF3 transfected cells.
As expected, the dominant active form of M-Ras bound more GTP than the
wild-type M-Ras.
View larger version (48K):
[in this window]
[in a new window]
| Fig 8.
Constitutive active M-Ras mutants bind GTP. Thin-layer
chromatography of in vivo 32P-labeled GDP and GTP eluted
from M-Ras immunoprecipitated from BAF-3 cells transfected with the
indicated experimental plasmids encoding the wild-type or activated
mutant M-Ras/22 Val. Same results were obtained with M-Ras/71Lys and
M-Ras/22Lys.
|
|
To gain a further understanding on the signal transduction pathway of
M-Ras, we tested the ability of our mutants to activate the MAPK in an
in vivo signal transduction pathway reporting system. This system
allows for the indirect measurement of the phosphorylation of a
chimeric transcriptional trans-activator consisting of the GAL4 DNA
binding domain fused to the activation domain of Elk (target of the
MAPK pathway; pFA-Elk). HEK293 cells were cotransfected with the
following 3 constructs: (1) a luciferase reporter gene (pFR-Luc)
controlled by a promoter containing 4 copies of a GAL4 binding motif,
(2) a plasmid inducing expression of the fusion protein mentioned
above, (3) a plasmid encoding the wild-type or mutated M-Ras. Lysates
of cells coexpressing GAL4-Elk fusion protein and the constitutively
activated MEK1 (used as positive control) contained high luciferase
activity (Fig 9A). Similarly, transfection
with mutant M-Ras/71K resulted in a 5.5-fold increase of Elk-mediated
transcription, compared with the empty vector or wild-type M-Ras. The
M-Ras-mediated transcriptional activation of Elk was blocked by the
MEK1 inhibitor PD9805929 (Fig 9B), while activation of the
JNK pathway by constitutively activated MEKK was unaffected even at the
highest concentration tested (80 µmol/L) (data not shown).
View larger version (14K):
[in this window]
[in a new window]
| Fig 9.
Activated M-Ras protein induces Elk-1-mediated
transcriptional activation. (A) HEK293 cells (5 × 105)
were transiently cotransfected by lipofection with 500 ng of pFR-Luc
reporter plasmid, 500 ng of the indicated experimental plasmids
encoding the wild-type or activated mutants M-Ras, and 25 ng of the
pathway-specific trans-activator plasmids (Gal4/Elk-1). pFC-MEK1
plasmid encodes constitutively activated MEK1 and was used as a
positive control for Elk-1-trans-activation, and pFC-dbd as a negative
control (Gal4). Luciferase activity was determined 24 hours after
transfection. Data represent the means ± SD from an experiment
performed in triplicate. The experiment was repeated 3 times with
similar results. (B) HEK293 cells (5 × 105) were
cotransfected with the reporter plasmid, pFR-Luc, the trans-activator
plasmid pFA-Elk-1 and an expression vector encoding activated
M-Ras/71K. After 5 hours of transfection, increasing amounts of PD98059
were added to the cell cultures and luciferase activity was measured
after 24 hours. Similar results were obtained with other activated
M-Ras mutants (data not shown). As a toxicity control, HEK293 cells
were transfected with the reporter plasmid, pFR-Luc, the
trans-activator plasmid pFA-Jun, and an expression vector encoding
activated MEKK with or without PD98059, and no inhibition was observed
(data not shown).
|
|
Activated M-Ras induces IL-3-independent proliferation of the mouse
pro-B lymphocyte cell line BaF3.
The M-Ras mutants were tested for their ability to support growth
factor-independent cell proliferation by transfecting BaF3 cells with
wild-type or dominant active forms of M-Ras. After selection in
puromycin, Western blot analysis of transfected BaF3 cells confirmed
comparable but elevated levels of M-Ras, while no expression was
detected in the parental, untransfected BaF3 cells
(Fig 10A). Parental BaF3 cells undergo
apoptosis in absence of IL-3 (data not shown). Overexpression of
wild-type M-Ras did not affect survival or proliferation of the
transfectant cells (Fig 10B). By contrast, M-Ras/22V, M-Ras/22K, or
M-Ras/71K-transfected BaF3 cells continued to proliferate in the
absence of IL-3. This proliferation does not appear to result from an
autocrine loop due to the activation of the IL-3 gene by M-Ras, because
the supernatant of M-Ras-transfected cells did not induce
proliferation of the parental BaF3 cells (data not shown). In addition,
IL-3 further increased the proliferation rate of BaF3 cells expressing
activated M-Ras, probably reflecting the fact that IL-3 induces an
additional growth-promoting pathway.30
View larger version (20K):
[in this window]
[in a new window]
| Fig 10.
Activated M-Ras induces cytokine-independent
proliferation. (A) BaF-3 transfected cells with empty vector (lane 1),
wild-type M-Ras (lane 2), constitutively activated M-Ras/22Val (lane
3), M-Ras/22Lys (lane 4), or M-Ras/71Lys (lane 5) were lysed and
analyzed by SDS-PAGE (14% acrylamide, 2 × 105 cells per
lane). M-Ras proteins were detected by immunoblotting. (B) Transfected
BaF3 cells (105 per well) were cultured with or without
IL-3 (200 U/mL). Cells were counted and sequentially diluted as
appropriate. The results represent the mean of 2 separate cultures that
varied by less than 10%.
|
|
To assess whether the MAPK pathway, which is induced by constitutive
M-RAS mutants in HEK293 cells (Fig 9), was responsible for this
M-Ras-induced proliferation, we treated BaF3 cells expressing M-Ras/71K with the MEK inhibitor PD98059. As shown in
Fig 11, the proliferation induced by
activated M-Ras was reduced to 12% by 20 µmol/L of PD98059 compared
with the untreated control. These data suggest that MAPK activation is
required for M-Ras-induced proliferation of BaF3 cells. By contrast,
in the presence of IL-3, the growth of these BaF3 cells was maintained
at about 90% of control, thus showing that the effect of PD98059 on
M-Ras-dependent proliferation was not due to nonspecific cytotoxicity
(Fig 11) and confirming that, in BaF3 cells, the MAPK pathway is
dispensable for IL-3-driven proliferation.30 The
observation that active M-Ras promoted survival and proliferation of
IL-3-starved pro-B lymphocytes is suggestive of the oncogenic activity
of these M-Ras mutations.
View larger version (15K):
[in this window]
[in a new window]
| Fig 11.
Inhibition of M-Ras-induced proliferation of BaF3 by
MAP kinase inhibitor. BaF3 cells expressing constitutively activated
M-Ras/71K (3,000/well) were incubated with increasing concentrations of
PD98059 in the presence or absence of 200 U/mL of IL-3. Thymidine
incorporation was measured after 3 days. The results are presented as
the percentage of the proliferation without PD98059 (means of
triplicate wells). The same results were obtained with other activated
M-Ras mutants (data not shown).
|
|
| |
DISCUSSION |
In an attempt to identify genes selectively induced by IL-9 in mouse
T-helper-cell clones, we found that IL-9 upregulates the expression of
M-Ras, the newest member of the Ras superfamily. This gene was cloned
independently by 2 other groups on the basis of sequence homologies
within this family of proteins and designated M-Ras or
R-Ras3.23,24 Interestingly, this induction by IL-9 seems to
depend on the activation of STAT transcription factors because a
mutated IL-9 receptor, which lacks STAT activity but is able to induce
proliferation and activation of various kinases (JAK-1, JAK-3, PI-3
kinase, etc),14 failed to mediate IL-9-induced M-Ras gene
expression. This observation, together with the rapid induction in
response to IL-9, suggests that M-Ras is regulated at the
transcriptional level. Because IL-2 activates STAT-5, and IL-9
activates STAT-1, -3, and -5, it is most likely that M-Ras gene
induction results from the activation of STAT-1 and/or STAT-3, as
recently observed for other IL-9-induced genes such as Granzyme A and
Ly6A2.31
Contrasting with the potent induction of M-Ras expression by IL-9, this
cytokine failed to induce M-Ras activation, as measured by its GTP/GDP
binding state. This suggests that M-Ras is not a direct component of
IL-9-dependent signal transduction pathways. However, we show here
that IL-3, a cytokine known to synergize with IL-9,12 is a
potent activator of M-Ras. Taken together, these observations point to
a role for IL-9 as competence factor, upregulating the ability of
effector cells to respond to Ras-activating cytokines. Interestingly,
other T-cell activators, such as Concanavalin A, similarly upregulate
M-Ras expression in mouse spleen cells, indicating that M-Ras
upregulation is not limited to the IL-9 response but takes part
in various T-cell activation processes.
M-Ras is closely related to R-Ras, TC21, H-Ras, K-Ras, and N-Ras, and
can therefore be considered as a member of the "true" Ras
subfamily. Members of the Ras subfamily are thought to control cell
growth, apoptosis, and transformation. Here, we studied the effect of
M-Ras activation on proliferation of BaF3 cells transfected with
constitutively activated mutants of M-Ras. Activated M-Ras was found to
mediate cytokine-independent survival and proliferation of the cells, a
result similar to the one reported for activated R-Ras that prevented
cell death of BaF3 cells caused by IL-3 deprivation.32 However, the activity of R-Ras in these cells was dependent on the
presence of serum or IGF-1, while, in our hands, M-Ras induced cell
survival even in the absence of serum (data not shown). The ability of
activated M-Ras to support the proliferation of BaF3 cells is likely to
be mediated by the MAPK pathway. A transient expression system using
HEK293 cells showed that M-Ras activates the MAPK pathway via the
activation of c-Raf (data not shown), which eventually results in
Elk-dependent transcriptional activation. Moreover, the M-Ras-induced
proliferation of BaF3 was blocked by the MEK inhibitor PD98059,
indicating that this function of M-Ras requires MAPK activity. This is
in agreement with the previous finding that activation of the Raf/MAPK
pathway in IL-3-dependent cells by expression of an oncogenic Raf or a
Ras mutant prevented apoptosis following IL-3
deprivation.33 The fact that aberrant M-Ras activation can
cause malignant transformation suggests that deregulation of M-Ras
function may also contribute to spontaneous malignancies, particularly
in IL-9 transgenic mice. In these mice, constitutive IL-9
overexpression results in a high susceptibility to the development of
T-cell lymphomas upon exposure to low doses of a chemical mutagen,
which was reported to induce Ras mutations.4 Interestingly,
the fact that M-Ras expression was also found in mitotically quiescent
tissues such as brain and kidney suggests that M-Ras plays a role in
other physiological processes as well.
To our knowledge, it is the first time that a cytokine has been shown
to induce the expression of a member of the Ras superfamily. In
contrast to H-, N-, or R-Ras, which are constitutively expressed in TS2
cells (data not shown), M-Ras is only expressed upon IL-9 stimulation.
Although IL-9 by itself cannot activate M-Ras, our observations
illustrate a new cross-talk mechanism between JAK/STAT and
MAPK-dependent pathways, which might explain, for instance, some of the
activities of IL-9 in synergy with other cytokines such as IL-3 or stem
cell factor.
| |
ACKNOWLEDGMENT |
The authors thank Drs S. Nagata, M. Gueguen, and A. Burgess for their
generous donations of reagents; Dr V. Stroobant for peptide synthesis;
and Dr M. Lackmann for helpful discussions and critical reading of the manuscript.
| |
FOOTNOTES |
Submitted February 23, 1999; accepted May 3, 1999.
Supported in part by the Belgian Federal Service for Scientific,
technical and cultural affairs and the Opération
Télévie. J.L. is a scientific associate
(Télévie) and J.-C.R. a research associate with the Fonds
National de la Recherche Scientifique, Belgium.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Jean-Christophe Renauld, MD,
Ludwig Institute for Cancer Research, Avenue Hippocrate, 74, UCL 7459, B-1200 Brussels, Belgium; e-mail: renauld{at}licr.ucl.ac.be.
| |
REFERENCES |
1.
Uyttenhove C, Simpson R, Van Snick J:
Functional and structural characterization of P40, a mouse glycoprotein with T cell growth factor activity.
Proc Natl Acad Sci USA
85:6934, 1988[Abstract/Free Full Text]
2.
Renauld J-C, Kermouni A, Vink A, Louahed J, Van Snick J:
Interleukin 9 and its receptor: Involvement in mast cell differentiation and T cell oncogenesis.
J Leukoc Biol
57:353, 1995[Abstract]
3.
Demoulin J-B, Renauld J-C:
Interleukin 9 and its receptor: An overview of structure and function.
Int Rev Immunol
16:345, 1998[Medline]
[Order article via Infotrieve]
4.
Renauld J-C, van der Lugt N, Vink A, van Roon M, Godfraind C, Warnier G, Merz H, Feller A, Berns A, Van Snick J:
Thymic lymphomas in interleukin 9 transgenic mice.
Oncogene
9:1327, 1994[Medline]
[Order article via Infotrieve]
5.
Merz H, Houssiau FA, Orscheschek K, Renauld J-C, Fliedner A, Herin M, Noel H, Kadin M, Mueller-Hermelink HK, Van Snick J, Feller AC:
IL-9 expression in human malignant lymphomas: Unique association with Hodgkin's disease and large cell anaplastic lymphoma.
Blood
78:1311, 1991[Abstract/Free Full Text]
6.
Nicolaides N, Holroyd KJ, Ewart SL, Eleff SM, Kiser MB, Dragwa CR, Sullivan CD, Grasso L, Zhang LY, Messler CJ, Zhou T, Kleeberger SR, Buetow KH, Levitt RC:
Interleukin 9: A candidate gene for asthma.
Proc Natl Acad Sci USA
94:13175, 1997[Abstract/Free Full Text]
7.
McLane MP, Haczku AH, van de Rijn M, Weiss C, Ferrante V, MacDonald D, Renauld JC, Nicolaides NC, Levitt RC:
Interleukin-9 promotes allergen-induced eosinophilic inflammation and airway hyperresponsiveness in transgenic mice.
Am J Resp Cell Mol
19:713, 1998[Abstract/Free Full Text]
8.
Temann UA, Geba GP, Rankin JA, Flavell RA:
Expression of Interleukin 9 in the lungs of transgenic mice causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness.
J Exp Med
188:1307, 1998[Abstract/Free Full Text]
9.
Holroyd KJ, Martinati LC, Trabetti E, Scherpbier T, Eleff SM, Boner AL, Pignatti PF, Kiser MB, Dragwa CR, Hubbard F, Sullivan CD, Grasso L, Messler CJ, Huang M, Hu Y, Nicolaides NC, Buetow KH, Levitt RC:
Asthma and bronchial hyperresponsiveness linked to the XY long arm pseudoautosomal region.
Genomics
52:233, 1998[Medline]
[Order article via Infotrieve]
10.
Petit-Frère C, Dugas B, Braquet P, Mencia-Huerta J-M:
Interleukin-9 potentiates the interleukin-4-induced IgE and IgG1 release from murine B lymphocytes.
Immunology
79:146, 1993[Medline]
[Order article via Infotrieve]
11.
Dugas B, Renauld J-C, Pène J, Bonnefoy JY, Petit-Frère C, Braquet P, Bousquet J, Van Snick J, Mencia-Huerta J-M:
Interleukin-9 potentiates the interleukin-4-induced immunoglobulin (IgG, IgM and IgE) production by normal human B lymphocytes.
Eur J Immunol
23:1687, 1993[Medline]
[Order article via Infotrieve]
12.
Hültner L, Druez C, Moeller J, Uyttenhove C, Schmitt E, Rüde E, Dörmer P, Van Snick J:
Mast cell growth enhancing activity (MEA) is structurally related and functionally identical to the novel mouse T cell growth factor P40/TCGFIII (interleukin-9).
Eur J Immunol
20:1413, 1990[Medline]
[Order article via Infotrieve]
13.
Louahed J, Kermouni A, Van Snick J, Renauld J-C:
IL-9 induces expression of granzymes and high affinity IgE receptor in murine T helper clones.
J Immunol
154:5061, 1995[Abstract]
14.
Demoulin J-B, Uyttenhove C, Van Roost E, de Lestré B, Donckers D, Van Snick J, Renauld J-C:
A single tyrosine of the Interleukin-9 (IL-9) receptor is required for STAT activation, antiapoptotic activity, and growth regulation by IL-9.
Mol Cell Biol
16:4710, 1996[Abstract]
15.
Palacios R, Steinmetz M:
IL3-dependent mouse clones that express B-220 surface antigen, contain Ig genes in germ-line configuration and generate B lymphocytes in vivo.
Cell
41:727, 1985[Medline]
[Order article via Infotrieve]
16.
Schmitt E, van Brandwijk R, Van Snick J, Siebold B, Rüde E:
TCGF III/P40 is produced by naive murine CD4+T cells but is not a general T cell growth factor.
Eur J Immunol
19:2167, 1989[Medline]
[Order article via Infotrieve]
17.
Druez C, Coulie P, Uyttenhove C, Van Snick J:
Functional and biochemical characterization of mouse P40/IL-9 receptors.
J Immunol
145:2494, 1990[Abstract]
18.
Ausubel F, Brent R, Kingston R, Moore D, Seidman J, Smith J, Struhl K:
Current Protocols in Molecular Biology, vol 1 and 2. New York, NY, Greene Publishing Association, 1993.
19.
Hubank M, Schatz DG:
Identifying differences in mRNA expression by representational difference analysis of cDNA.
Nucleic Acids Res
22:5640, 1994[Abstract/Free Full Text]
20.
Schreiber E, Matthias P, Müller M, Schaffner W:
Rapid detection of octamer binding proteins with "mini-extracts", prepared from a small number of cells.
Nucleic Acids Res
17:6419, 1989[Free Full Text]
21.
Mizushima S, Nagata S:
pEF-BOS, a powerful mammalian expression vector.
Nucleic Acids Res
18:5322, 1990[Free Full Text]
22.
Louahed J, Renauld J-C, Demoulin J-B, Baughman G, Bourgeois S, Sugamura K, Van Snick J:
Differential activity of dexamethasone on IL-2-, IL-4-, or IL-9-induced proliferation of murine factor-dependent T cell lines.
J Immunol
156:3704, 1996[Abstract]
23.
Kimmelman A, Tolkacheva T, Lorenzi MV, Osada M, Chan AM-L:
Identification and characterization of R-ras3: A novel member of the RAS gene family with a non-ubiquitous pattern of tissue distribution.
Oncogene
15:2675, 1997[Medline]
[Order article via Infotrieve]
24.
Matsumoto K, Asano T, Endo T:
Novel small GTPase M-Ras participates in reorganization of actin cytoskeleton.
Oncogene
15:2409, 1997[Medline]
[Order article via Infotrieve]
25.
Glomset JA, Farnsworth CC:
Role of protein modification reactions in programming interactions between ras-related GTPases and cell membranes.
Annu Rev Cell Biol
10:181, 1994
26.
Gelb MH:
Protein prenylation, et cetera: Signal transduction in two dimensions.
Science
275:1750, 1997[Free Full Text]
27.
Godfraind C, Louahed J, Faulkner H, Vink A, Warnier G, Grencis R, Renauld J-C:
Intraepithelial infiltration by mast cells with both connective tissue-type and mucosal-type characteristics in gut, trachea, and kidneys of IL-9 transgenic mice.
J Immunol
160:3989, 1998[Abstract/Free Full Text]
28.
Feig L, Cooper GM:
Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins.
Mol Cell Biol
8:2472, 1988[Abstract/Free Full Text]
29.
Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR:
PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo.
J Biol Chem
270:27489, 1995[Abstract/Free Full Text]
30.
Terada K, Kaziro Y, Satoh T:
Ras is not required for interleukin 3-induced proliferation of a mouse pro-B cell line, BaF3.
J Biol Chem
270:27880, 1995[Abstract/Free Full Text]
31.
Demoulin JB, Maisin D, Renauld J-C:
Ly-6A/E induction by interleukin-6 and interleukin-9 in T cells.
Eur Cytokine Netw
10:49, 1999[Medline]
[Order article via Infotrieve]
32.
Suzuki J, Kaziro Y, Koide H:
An activated mutant of R-Ras inhibits cell death caused by cytokine deprivation in BaF3 cells in the presence of IGF-I.
Oncogene
15:1689, 1997[Medline]
[Order article via Infotrieve]
33.
Kinoshita T, Shirouzu M, Kamiya A, Hashimoto K, Yokoyama S, Miyajima A:
Raf/MAPK and rapamycin-sensitive pathways mediate the anti-apoptotic function of p21Ras in IL-3-dependent hematopoietic cells.
Oncogene
15:619, 1997[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
N. Nunez Rodriguez, I. N. L. Lee, A. Banno, H. F. Qiao, R. F. Qiao, Z. Yao, T. Hoang, A. C. Kimmelman, and A. M.-L. Chan
Characterization of R-ras3/m-ras null mice reveals a potential role in trophic factor signaling.
Mol. Cell. Biol.,
October 1, 2006;
26(19):
7145 - 7154.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Sun, H. Watanabe, K. Takano, T. Yokoyama, J.-i. Fujisawa, and T. Endo
Sustained activation of M-Ras induced by nerve growth factor is essential for neuronal differentiation of PC12 cells.
Genes Cells,
September 1, 2006;
11(9):
1097 - 1113.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Lionikas, D. A. Blizard, G. S. Gerhard, D. J. Vandenbergh, J. T. Stout, G. P. Vogler, G. E. McClearn, and L. Larsson
Genetic determinants of weight of fast- and slow-twitch skeletal muscle in 500-day-old mice of the C57BL/6J and DBA/2J lineage
Physiol Genomics,
April 14, 2005;
21(2):
184 - 192.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Chiara, S. Bishayee, C.-H. Heldin, and J.-B. Demoulin
Autoinhibition of the Platelet-derived Growth Factor {beta}-Receptor Tyrosine Kinase by Its C-terminal Tail
J. Biol. Chem.,
May 7, 2004;
279(19):
19732 - 19738.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K.-X. Zhang, K. R. Ward, and J. W. Schrader
Multiple Aspects of the Phenotype of Mammary Epithelial Cells Transformed by Expression of Activated M-Ras Depend on an Autocrine Mechanism Mediated by Hepatocyte Growth Factor/Scatter Factor
Mol. Cancer Res.,
April 1, 2004;
2(4):
242 - 255.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Baraldo, D. S. Faffe, P. E. Moore, T. Whitehead, M. McKenna, E. S. Silverman, R. A. Panettieri Jr., and S. A. Shore
Interleukin-9 influences chemokine release in airway smooth muscle: role of ERK
Am J Physiol Lung Cell Mol Physiol,
June 1, 2003;
284(6):
L1093 - L1102.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. C. Kimmelman, N. N. Rodriguez, and A. M.-L. Chan
R-Ras3/M-Ras Induces Neuronal Differentiation of PC12 Cells through Cell-Type-Specific Activation of the Mitogen-Activated Protein Kinase Cascade
Mol. Cell. Biol.,
August 15, 2002;
22(16):
5946 - 5961.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-B. Demoulin, C. Uyttenhove, D. Lejeune, A. Mui, B. Groner, and J.-C. Renauld
STAT5 Activation Is Required for Interleukin-9-dependent Growth and Transformation of Lymphoid Cells
Cancer Res.,
July 1, 2000;
60(14):
3971 - 3977.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
L. Dumoutier, J. Louahed, and J.-C. Renauld
Cloning and Characterization of IL-10-Related T Cell-Derived Inducible Factor (IL-TIF), a Novel Cytokine Structurally Related to IL-10 and Inducible by IL-9
J. Immunol.,
February 15, 2000;
164(4):
1814 - 1819.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Ohba, N. Mochizuki, S. Yamashita, A. M. Chan, J. W. Schrader, S. Hattori, K. Nagashima, and M. Matsuda
Regulatory Proteins of R-Ras, TC21/R-Ras2, and M-Ras/R-Ras3
J. Biol. Chem.,
June 23, 2000;
275(26):
20020 - 20026.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. F. Rebhun, A. F. Castro, and L. A. Quilliam
Identification of Guanine Nucleotide Exchange Factors (GEFs) for the Rap1 GTPase. REGULATION OF MR-GEF BY M-Ras-GTP INTERACTION
J. Biol. Chem.,
November 3, 2000;
275(45):
34901 - 34908.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION