From the Department of Biochemistry, American Red
Cross, Rockville, Maryland 20855, the ¶ Institute of Biology,
University of Palermo, 90133 Palermo, and the Department of Biology,
Genetics and Medical Chemistry, University of Torino,
10126 Torino, Italy
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ABSTRACT |
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Integrins are heterodimeric transmembrane
receptors involved in the regulation of cell growth and
differentiation. The
1 integrin subunit is widely
expressed in vivo and is represented by four alternatively
spliced cytoplasmic domain isoforms.
1D is a
muscle-specific variant of
1 integrin and a predominant
1 isoform in striated muscles. In the present study we
showed that expression of the exogenous
1D integrin in
C2C12 myoblasts and NIH 3T3 or REF 52 fibroblasts inhibited cell
proliferation. Unlike the case of the common
1A isoform,
adhesion of
1D-transfected C2C12 myoblasts specifically
via the expressed integrin did not activate mitogen-activated protein
kinases. The
1D-induced growth inhibitory signal was
shown to occur late in the G1 phase of the cell cycle,
before the G1-S transition. Ha-(12R)Ras, but not
(
22W)Raf-1 oncogene, was able to overcome completely the
1D-triggered cell growth arrest in NIH 3T3 fibroblasts.
Since perturbation of the
1D amino acid sequence in
1A/
1D chimeric integrins decreased the
growth inhibitory signal, the entire cytoplasmic domain of
1D appeared to be important for this function. However,
an interleukin-2 receptor-
1D chimera containing the
cytoplasmic domain of
1D still efficiently inhibited
cell growth, showing that the ectodomain and the ligand-binding site in
1D were not required for the growth inhibitory signal.
Together, our data showed a new specific function for the alternatively
spliced
1D integrin isoform. Since the onset of
1D expression during myodifferentiation coincides with the timing of myoblast withdrawal from the cell cycle, the growth inhibitory properties of
1D demonstrated in this study
might reflect the major function for this integrin in commitment of differentiating skeletal muscle cells in vivo.
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INTRODUCTION |
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Integrins are a large family of heterodimeric transmembrane cell
adhesion receptors (1). They are involved in many aspects of cell
behavior and are known to regulate a number of intracellular signaling
pathways (2-4). One of the key integrin functions is an
adhesion-mediated growth signaling (5). Integrin-mediated positive cell
growth signaling cascade leads to activation of MAP1 kinases, which serves as
a hallmark of cell proliferation (6-9). Integrins can synergize with
growth factor receptors to relay the control of cell cycle progression
in adhesion-dependent cell types (5, 10). Adhesion of
different cell types to certain ECM proteins can either trigger
proliferation or switch them to a differentiation program (11, 12).
These opposite responses are possible due to redundant and overlapping
expression of integrins and are mediated by ligation of different
integrin heterodimers on the cell surface (1). Differential
coupling of the Shc adapter protein to integrin
subunits causes
selective activation of the MAP kinase cascade in adherent cells (13).
This allows various integrin
heterodimers to transduce distinct
signals from the ECM to the cell interior, including positive and
negative growth signals in response to adhesion.
1 integrin, the most ubiquitous
subunit, pairs with
at least 10 different
subunits to comprise receptors for a wide
variety of ECM proteins. It is abundantly expressed in vivo
on all proliferating as well as differentiated growth-arrested cell
types, excluding red blood cells (1).
1 integrin is
known to be involved in cell growth regulation in many cell types (14,
15). Four cytoplasmic domain variants generated by alternative splicing
have been described for the
1 subunit (Refs. 16-21 and
reviewed in Ref. 22). In most cell types
1 integrin is
represented predominantly by the
1A isoform. The only
noticeable exception is in differentiated striated muscles where it is
displaced by the
1D integrin isoform (16). The other two
minor
1 integrin variants with the alternatively spliced
cytoplasmic domains,
1B and
1C, were
identified several years ago (18-21). Unlike
1D
integrin, these two isoforms are always coexpressed at low levels with
the major
1A isoform (18, 19, 21). Recently, the
1C isoform has been shown to inhibit strongly cell
growth upon transient expression in 10T1/2 fibroblasts (23). A short
amino acid sequence Gln795-Gln802 within the
cytoplasmic domain is essential for
1C-mediated growth arrest and is apparently unique for this integrin (24).
Striated muscle myoblasts become irreversibly withdrawn from the cell
cycle early in myodifferentiation before cell fusion occurs. The timing
of cell cycle withdrawal (commitment to myodifferentiation) coincides
with the appearance of muscle-specific 1D integrin in
myocyte cultures and is in accordance with its increased expression in
postmitotic growth-arrested myoblasts (16). We hypothesized that
1D might transmit a growth inhibitory signal in normal
myoblasts in vivo. Here we demonstrate an inhibition of cell
proliferation by
1D integrin when it is expressed
transiently in normal myoblasts and fibroblasts.
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MATERIALS AND METHODS |
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Antibodies and Reagents--
Thymidine analog bromodeoxyuridine
(BrdUrd) and mouse anti-BrdUrd mAb were from Sigma. Mouse mAb TS2/16
against human 1 integrin was provided by Dr. Martin
Hemler (Dana Farber Cancer Institute, Boston) and used to detect the
injected or transfected
1A and
1D
integrins. Hamster anti-mouse
1 mAb HM
1-1 for
visualization of the endogenous
1 integrin was received
from PharMingen (San Diego, CA). Anti-MAP kinase rabbit polyclonal
antibody sc-93 was obtained from Santa Cruz Biotechnology
(Santa Cruz, CA) and was used for MAP kinase immunoprecipitation.
Antibody against active (dually phosphorylated) MAP kinases was from
Promega (Madison, WI). Rabbit polyclonal antibodies reacting with mouse
cyclins A and E were obtained from Rockland (Gilbertsville, PA). Mouse mAb 7G7B6 against interleukin-2 receptor (IL2R) extracellular domain
was from ATCC (Rockville, MD). Hamster mAbs reacting with mouse
1,
2, and
5 integrins were
obtained from PharMingen. Antibody against
3 integrin
cytoplasmic domain was kindly provided by Dr. Guido Tarone (University
of Torino, Italy). Isoform-specific anti-
1A and
anti-
1D antibodies were characterized previously (16).
Cell Cultures--
Mouse C2C12 myoblasts and NIH 3T3 fibroblasts
were obtained from ATCC and used between 3 and 10 passages.
Ha-(12R)Ras- and (22W)Raf-transfected NIH 3T3 fibroblasts were
described previously (25-28). Briefly, activated Ha-(12R)Ras and
activated (
22W)Raf-1 (25, 26) were cloned into the expression vector
pZIP-NeoSV(×)1 and transfected into NIH 3T3 cells using the calcium
phosphate precipitation technique essentially as reported previously
(25, 27). 24 h after transfection G418 was added to the growth
medium at 1 mg/ml. The cells were selected for 10 days by which time all the cells in a control mock-transfected dish were dead. NIH 3T3
cells stably expressing Ha-(12R)Ras or (
22W)Raf-1 displayed very
similar highly transformed phenotype as detected by cell morphology,
inhibited spreading, colony formation in soft agar, and profound
cytoskeletal changes (26, 28). Rat embryo fibroblast line REF 52 was
described previously (29).
1A-CHO and
1D-CHO stable transfectants were characterized
previously (16, 17).
cDNA Constructs, Microinjection, and Transfection--
The
cDNAs for human 1A and
1D cytoplasmic
domain isoforms in pECE vector were described previously (16, 19).
cDNAs encoding
1A/
1D chimeras were
prepared using the
1A and
1D cDNA
fragments and polymerase chain reaction-based mutagenesis. The
sequences of the four mutant
1A/
1D
integrin cDNAs were verified by dideoxy termination sequencing.
IL2R (interleukin 2 receptor) cDNA and IL2R-
1A
chimera (30) were kindly provided by Dr. Susan LaFlamme (Albany Medical
College, Albany, NY). IL2R-
1D chimera was generated using polymerase chain reaction amplification of the 3' end of the
1D cDNA (16) with the forward primer
5'-TGTAGCTGGTGTGGTTGCTG-3' and the reverse primer
5'-TTCAAAGCTATTCTGGGCTG-3'. The polymerase chain reaction fragment was
digested with HindIII endonuclease, and the resulting
fragment encoding the
1D cytoplasmic domain was inserted
in the HindIII site of the IL2R-
1A plasmid
(30) to generate a plasmid encoding the IL2R-
1D chimera.
The structure of the IL2R-
1D construct was confirmed by
dideoxynucleotide sequence analysis using a primer upstream of the
cloning site (5'-AGCGTCCTCCTCCTGAGTG-3').
Integrin Analysis by Flow Cytometry--
The expression levels
of the transfected human 1A and
1D
integrins were determined using fluorescence-activated cell sorter analysis with TS2/16 mAb which reacts identically with these two
1 cytoplasmic domain variants (16, 17). The
transfectants were incubated with 10 µg/ml of TS2/16 mAb for 1 h
at 4 °C followed by fluorescein-labeled goat anti-mouse IgG. After
the staining, cells were analyzed in a FACScan® flow cytometer (Becton
Dickinson, Mountain View, CA).
Cell Proliferation Assay--
The cell proliferation assay based
on BrdUrd incorporation into the nuclei was described earlier (23, 31).
Cells transfected with 1A,
1D cDNAs,
1A/
1D chimeras, or IL2R-
1A
and IL2R-
1D constructs were trypsinized on the day after
transfection and were plated on fibronectin-coated glass coverslips in
DMEM containing 10% FBS. The next day, the growth medium was replaced
for DMEM with 0.5% FBS and cells were kept in low serum for 24 h.
After that, cells were switched to the medium containing 10% FBS and 50 µM BrdUrd. 24 h later, cells were fixed with
3.7% formaldehyde in phosphate-buffered saline and permeabilized with
0.5% Triton X-100. Before the immunostaining, cells were incubated
first with DNase (0.1 units/µl, Promega) for 30 min at 37 °C.
Immunostaining of the Transfected and Injected Cells--
To
visualize simultaneously the transfected (injected) 1
integrins and nuclear incorporation of BrdUrd, fixed, permeabilized, and DNase-treated cells were coincubated with mAb TS2/16 against human
1 integrins and anti-BrdUrd mAb (1:100 dilution) for
1 h at 37 °C. Rhodamine-labeled donkey anti-mouse IgG
(Chemicon, Temecula, CA) was used as secondary antibody to visualize
the expressed
1 integrins at focal adhesions and
accumulation of BrdUrd in the nuclei. Coverslips were viewed on a Zeiss
Axiophot microscope equipped for epifluorescence. Micrographs were
taken on T-max 400 film. 200 untransfected cells or cells expressing
1A,
1D, or
1A/
1D chimeras were counted and the
nuclear staining for BrdUrd assessed.
MAP Kinase Assays--
Activation of MAP kinases in
1A- and
1D-transfected C2C12 cells was
studied as reported earlier (6, 9, 10, 16, 32, 33), with some
modifications. Briefly, cells transfected with human
1A
and
1D cDNAs were trypsinized on the day after transfection. The remaining trypsin was inhibited with 0.5 mg/ml soybean trypsin inhibitor, and cells were washed several times in
serum-free medium. Cells in DMEM, containing 2% bovine serum albumin,
were plated on bacterial Petri dishes, precoated with purified TS2/16
mAb against the transfected
1 integrins, for 2 h at
37 °C. Unbound cells were washed out, and the adherent cells
expressing human
1A or
1D integrins were
lysed either in 1% SDS or in buffer containing 1% Nonidet P-40, 0.5%
sodium deoxycholate, 150 mM NaCl, 50 mM HEPES,
pH 7.5, with 1 mM sodium orthovanadate, 50 mM
NaF, 1 mM p-nitrophenyl phosphate, 20 nM calyculin A, 10 µg/ml aprotinin, 10 µg/ml leupeptin,
and 1 mM phenylmethylsulfonyl fluoride. Protein
concentration in the samples was determined using Pierce BCA Protein
Assay Reagent. Samples in 1% SDS (25 µg each) were used directly for
SDS-polyacrylamide gel electrophoresis and subsequent immunoblotting
with polyclonal antibody against activated (dually phosphorylated) MAP
kinases (Promega). 0.5 mg of cell lysates in 1% Nonidet P-40, 0.5%
sodium deoxycholate buffer were used for immunoprecipitation with
sc-93 anti-MAP kinase antibody (Santa Cruz Biotechnology)
followed by immune kinase reaction with [
-32P]ATP and
the exogenous substrate myelin basic protein (MBP). Phosphorylated MBP
bands were analyzed by SDS electrophoresis and autoradiography (6,
16).
Metabolic Labeling, Immunoprecipitation, and
Immunoblotting--
Cultured NIH 3T3 fibroblasts (wild-type or
expressing exogenous Ha-(12R)Ras) were metabolically labeled with 0.1 mCi/ml Tran35S-label (ICN Biomedicals, Irvine, CA) in
methionine- and cysteine-free medium for 12 h at 37 °C. After
the labeling, cells were washed 3 times with phosphate-buffered saline
and lysed on ice for 3 min with RIPA buffer (150 mM NaCl,
50 mM Tris-Cl, pH 7.5; containing 0.1% SDS, 1% Triton
X-100 and 0.5% sodium deoxycholate). 1,
2,
3,
5, and the
endogenous mouse
1 integrins were immunoprecipitated using the specific antibodies against these subunits. The resulting immunoprecipitates were analyzed by SDS-polyacrylamide gel
electrophoresis on 10% gels.
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RESULTS |
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In our preliminary experiments we were not able to obtain stable
expression of 1D in normal myoblasts, including mouse
C2C12 cells or normal fibroblasts, such as REF 52 or NIH 3T3 cell
lines, suggesting that this integrin might directly affect cell growth. To examine this possibility, we first microinjected either
1A or
1D cDNAs into the nuclei of
these cells. The injected cells were serum-starved, then treated for
24 h with BrdUrd in serum-containing medium, and finally stained
for both human
1 integrin and BrdUrd incorporation (Fig.
1). No nuclear staining was seen in
1A- and
1D-injected cells with
anti-
1 integrin TS2/16 mAb alone (not shown). Whereas
the majority of untransfected and
1A-transfected cells
displayed bright nuclear staining with anti-BrdUrd mAb (Fig. 1,
A and C, arrows), most
1D-transfected C2C12, REF 52, and NIH 3T3 cells did not
incorporate BrdUrd into the nuclei (Fig. 1, B, D,
and E, arrowheads). This indicated that
1D caused growth arrest in these cell types. In the
injected cells,
1D colocalized with the endogenous
1A integrin at focal adhesions but did not cause a
displacement or relocalization of
1A from these sites (Fig. 1, E and F). Therefore, we concluded that
the observed inhibition of cell proliferation in
1D-transfected cells was caused by the expression of
1D integrin rather than the lack of the common
1A isoform at cell-matrix contacts.
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Transient transfection of 1A and
1D
cDNAs in C2C12, REF 52, and NIH 3T3 cells resulted in similar
expression levels of these integrins on the cell surface as determined
by flow cytometry with TS2/16 mAb (Table
I). Quantitation of the growth inhibitory effect mediated by
1D in the transfectants showed that
in the presence of this integrin only ~20% of C2C12 myoblasts (Fig.
2A) and ~14% of REF 52 fibroblasts (Fig. 2B) were entering the S phase, whereas
more than 75% of
1A transfectants were progressing into the cell cycle under conditions of this assay. Because the efficiency of cell growth arrest depends on the level of the exogenous
1D, the few proliferating
1D-transfected
cells might represent a subpopulation of the transfectants with the
lower level of
1D expression. These results show that
proliferation of normal myoblasts and fibroblasts is drastically
inhibited by
1D integrin.
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Analysis of MAP kinase activation in 1A- and
1D-transfected C2C12 cells was first performed in the
absence of exogenous growth factors in cells adherent to the substrate
specifically via the expressed integrin (Fig.
3, A and D).
1A-expressing cells displayed high levels of
adhesion-dependent MAP kinase activation measured by both
immunoblotting and immune complex kinase assays (Fig. 3, A
and D; a and a'). In contrast, MAP
kinase activity in the adherent
1D transfectants did not
differ from the basal levels characteristic for these cells in
suspension (Fig. 3, A and D; b and
b'). Unlike C2C12 cells expressing
1D,
1D-CHO transfectants exhibited sharp activation of MAP
kinases in response to
1D-mediated adhesion under the
same experimental conditions (Fig. 3, B and E and
Ref. 16). Therefore, the growth inhibitory effect of
1D strongly depends on cellular context and might be suppressed by enhanced activity of certain oncogenes.
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In the presence of growth factors both 1A- and
1D-transfected C2C12 cells displayed higher levels of
MAP kinase activation compared with serum-free conditions (Fig. 3,
C and F). However, whereas MAP kinase activation
further increased upon adhesion in
1A-transfected cells
(Fig. 3, C and F; a and
a'), no such enhancement was seen in
1D-expressing cells in the presence of serum (Fig. 3,
C and F; b and b'). This
observation showed that
1D down-regulates MAP kinase
activity in growth factor-stimulated C2C12 cells as well. Thus, the
withdrawal of differentiating myoblasts from the cell cycle in
vivo could be attributed at least partly to growth inhibitory
effect of
1D integrin.
To determine the phase of the cell cycle affected by 1D
expression, we double-stained both populations of NIH 3T3 transfectants for the expressed human
1 integrin and either cyclin E
or cyclin A (Fig. 4A). Scoring
the
1A- and
1D-transfected cells for
nuclear cyclin staining demonstrated that cyclin E expression did not significantly differ between the two populations. However, the percentage of
1A transfectants expressing cyclin A
exceeded drastically that of
1D-expressing cells. Since
the expression of cyclin A occurs specifically during the S phase and
1D blocks its appearance in the transfected NIH 3T3
cells, we concluded that
1D strongly interferes with the
G1-S transition. To analyze further the timing of the
1D-triggered cell cycle block, we microinjected
1D cDNA at certain time points after serum
stimulation of starved cells (Fig. 4B). DNA synthesis in NIH
3T3 cells became insensitive to inhibition by
1D
integrin when its cDNA was injected ~12 h after serum
stimulation. Previously, our estimations showed that the G1
phase lasts ~14-16 h in this cell type. Together, these two observations demonstrated that
1D-mediated growth arrest
occurs late in the G1 phase before the beginning of the S
phase.
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Previously we were able to select stable 1D integrin
transfectants of CHO cells (16) and GD25
1
integrin-deficient cells (17, 34), indicating that some cells were able
to proliferate in the presence of
1D. Since both these
cell lines display elevated activity of certain oncogenes, we
rationalized that some of them can overcome the
1D-triggered cell cycle block. To test this suggestion
directly, we transiently transfected
1D cDNA into NIH 3T3 cells expressing constitutively activated forms of Ha-Ras and
Raf-1 oncogenes (25-28). Analysis of cell proliferation by the BrdUrd
incorporation assay demonstrated that Ha-(12R)Ras completely abolished
the inhibitory effect of
1D on cell growth (Fig.
5). However, (
22W)Raf-1 was not able
even to diminish this
1D-mediated effect. These data
showed that certain oncogenes, such as Ha-Ras, overcome the
1D-mediated growth arrest, whereas some others, like
Raf-1, cannot.
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Since integrin subunits are known to modulate cell growth through
differential activation of MAP kinases (13), we asked whether activated
Ha-Ras could alter the expression pattern of
subunits in NIH 3T3
transfectants or their association with
1D integrin
(Fig. 6). We did not see any detectable
changes in the pattern of
subunits associated with
1
integrin in NIH 3T3 cells transfected with Ha-(12R)Ras compared with
the wild-type cells (Fig. 6, A and B).
3 and
5 integrins were the major
1-associated
subunits in this cell line. The
association of
1A and
1D integrins with
the
subunits was not altered in the corresponding transfectants (Fig. 6, C and D).
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We also wanted to examine which amino acid sequences in
1D integrin are required and sufficient for its growth
inhibitory function. We therefore created
1 integrin
chimeras by swapping
1A and
1D
cytoplasmic domain sequences (Table II).
All of these chimeric
1A/
1D integrins
were expressed at similar levels on the cell surface and were targeted
efficiently to focal adhesions of C2C12 cells, with the exception of
chimera 4. None of the C2C12 transfectants expressing these chimeric
integrins displayed abnormal adhesion or spreading (not shown).
Interestingly, when examined by cell proliferation assay, none of the
chimeras was as efficient as
1D with regard to growth
inhibition (Fig. 7A). The only
chimera that still caused a significant decrease of C2C12 cell
proliferation was chimera 2, which lacked only 6 amino acids at the
very C terminus of the
1D polypeptide (Fig.
7A, Table II). These data strongly suggest that there is no
short amino acid motif within the
1D cytoplasmic tail,
which is sufficient for this function. Instead, the whole C-terminal
half of the cytodomain, encoded by exon D of the
1
integrin gene (16), appears to be essential for cell growth arrest.
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By trying to determine whether the extracellular domain and ligand
binding have a role in the growth inhibitory function of 1D integrin, we took advantage of earlier engineered
chimeric receptors, such as the IL2R-
1A chimera (30).
This chimera contains the extracellular and transmembrane domains of
interleukin 2 receptor and the cytoplasmic domain of
1A
integrin. By replacing the
1A portion of this construct
with the cDNA fragment encoding the
1D cytoplasmic
domain (see "Materials and Methods"), we generated IL2R-
1D chimeric receptor. Both IL2R-
1D
and IL2R-
1A chimeras were similarly expressed on the
cell surface (Table III) and were targeted to focal adhesions upon transient expression in C2C12 cells
(data not shown). The expression of these two chimeras as well as the
wild-type IL2R in C2C12 myoblasts was followed by the analysis of cell
proliferation using BrdUrd incorporation method (Fig. 7B).
Our results showed that IL2R-
1A chimera only slightly
decreased proliferation, most likely due to partial disruption of focal
adhesions in some of the transfectants expressing high levels of this
construct. In contrast, IL2R-
1D chimeric receptor strongly inhibited cell cycle progression in C2C12 myoblasts (Fig. 7B).
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DISCUSSION |
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In this work we demonstrated that 1D, the
muscle-specific variant of the
1 integrin subunit,
inhibits cell cycle progression in normal myoblasts and fibroblasts.
Our data reveal a novel function for the
1D cytoplasmic
domain isoform, which is the predominant
1 integrin in
striated muscles (16). This growth inhibitory function is specifically
ascribable to the alternatively spliced cytoplasmic tail of
1D, since the entire cytodomain appears to be both
necessary and sufficient for the
1D-mediated growth
arrest.
1D exhibits a dominant-positive effect over the
1A isoform due to enhanced interactions with both
cytoskeletal and extracellular ligands and displaces the endogenous
1A from cell-matrix contacts when expressed in certain
cell types (17). However, in C2C12, REF 52 or NIH 3T3 cells expressing
high levels of
1D integrin, adhesion, and spreading on
various ECM proteins was not visibly affected. Also, our recent data on
transiently transfected
1D-C2C12 and
1D-NIH 3T3 cells indicated that even the cells
expressing low amounts of
1D and still having the
endogenous
1A at focal adhesions (Fig. 1, E
and F) were unable to proliferate. These observations
suggested that the
1D-triggered cell cycle block is not
caused by the lack of
1A at cell-matrix adhesions and consequent loss of
1A-mediated signaling but is
primarily due to active signaling by
1D. Meanwhile,
further analysis is needed to elucidate whether this effect is based on
the active inhibitory signal by
1D or blocking positive
adhesion-mediated signaling caused by this integrin variant.
Recently, another 1 integrin isoform,
1C,
was shown to generate a strong growth inhibitory signal upon its
transient expression in 10T1/2 fibroblasts (23) or CHO cells (24).
Expression of this integrin in vivo is detectable in some
blood cells (21) and is up-regulated in tumor necrosis
factor-
-stimulated endothelial cells (24). No information is
available yet concerning the mechanisms of growth arrest mediated by
1D and
1C isoforms. In both these cases,
the cytoplasmic domains alone are sufficient to generate the inhibitory
signal, whereas the transmembrane and extracellular domains, and
therefore ligand binding, are not required. However, in the case of
1C integrin, a short specific motif
Gln795-Gln802 is responsible for this function
(24), whereas the entire cytodomain appears to be important for the
growth arrest triggered by
1D. Another distinction
between the mechanisms of growth inhibition by these integrins relates
to the fact that
1C displays its antiproliferative potential in all the cell types tested so far (23, 24), although
1D growth inhibitory effect is clearly cell
type-specific.
Certain oncogenes, as shown here for Ha-(12R)Ras, are able to
completely suppress the 1D-mediated growth arrest. This
explains why we previously were able to obtain stable expression of
1D integrin in some cell types that had elevated
activity of some oncogenes (CHO and GD25
1-minus cells)
but failed to select stable transfectants with other cells displaying a
more normal phenotype. This property of
1D is also
consistent with the fact that some transformed cells of muscle origin,
e.g. human rhabdomyosarcoma (RD) cells, proliferate even
though expressing significant amounts of
1D
integrin.2 Yet, others have
shown that expression of c-myc and v-myc
oncogenes in C2C12 myocytes does not significantly alter either their
differentiation patterns or commitment (irreversible withdrawal from
the cell cycle), (35). Taken together, these observations show that the growth inhibitory effect of
1D can be suppressed by some
but not all oncogenes.
At present it remains unclear why activated Ha-Ras is able to overcome
the growth inhibitory signal from 1D whereas activated Raf-1 can not. Most existing models of anchorage-dependent
cell growth imply that Ras is positioned upstream from Raf-1 in the integrin-mediated MAP kinase cascade. Also, activated Raf-1 and Ha-Ras
have similar transforming activity in NIH 3T3 fibroblasts as judged by
cell morphology, growth in soft agar and cytoskeletal changes (26, 28).
However, one recent report showed that integrin-mediated activation of
MAP kinases, MEK (MAP kinase kinase) and Raf-1 in NIH 3T3 cells
appeared to be independent of Ras (33). Also, activation of the
Raf-1/MAP kinase cascade was shown to be insufficient for Ras
transformation of RIE-1 epithelial cells (26). These recent
observations imply that oncogenic Ras triggers certain Raf-independent
signals essential for cellular transformation and also reevaluate the
roles of Ras and Raf in propagation of adhesion-mediated growth signals
from integrins to MAP kinases.
Previous analysis of 1D expression in mouse myogenic
cultures (16) and during mouse
embryogenesis3 showed that
the onset of its biosynthesis coincides with the time point of
irreversible growth arrest during myocyte differentiation. This
indicates that the growth inhibitory properties of
1D,
demonstrated by transient transfection assays in this study, might be
related to the major function of this muscle-specific integrin in
vivo. Further work is apparently needed to elucidate the role of
specific signaling events in the cell growth arrest mediated by
1D integrin.
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ACKNOWLEDGEMENTS |
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We thank Dr. Keith Burridge (University of
North Carolina, Chapel Hill), in whose lab a part of this work was
performed, for support and encouragement. We are grateful to Drs. Guido
Tarone and Fiorella Balzac (University of Torino, Italy) for providing us with cDNAs encoding 1A/
1D integrin
chimeras. Dr. Susan LaFlamme (Albany Medical College, Albany, NY)
kindly supplied IL2R and IL2R-
1A constructs used in this
study. We are indebted to Drs. Geoffrey Clark and Channing Der
(University of North Carolina, Chapel Hill, NC) for providing us with
NIH 3T3 cells expressing Ha-(12R)Ras and (
22W)Raf-1 oncogenes. We
also thank Dr. Kenneth Ingham (Biochemistry Department, American Red
Cross) for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health R29 Grant CA77697 (to A. M. B.) and National Institutes of Health Grant GM29860 (to Dr. Keith Burridge).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.
§ To whom correspondence should be addressed: Dept. of Biochemistry, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0725; Fax: 301-738-0794; E-mail: belkina{at}usa.redcross.org.
1 The abbreviations used are: MAP, mitogen-activated protein; ECM, extracellular matrix; BrdUrd, bromodeoxyuridine; MBP, myelin basic protein; mAb, monoclonal antibody; IL2R, interleukin-2 receptor; DMEM, Dulbecco's modified Eagle's medium; CHO, Chinese hamster ovary; FBS, fetal bovine serum.
3 M. Brancaccio, S. Cabodi, A. M. Belkin, G. Collo, V. E. Koteliansky, D. Tomatis, F. Altruda, L. Silengo, and G. Tarone, manuscript in preparation.
2 A. M. Belkin, unpublished observations.
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