(Received for publication, July 5, 1995; and in revised form, August 8, 1995)
From the
Alternative splicing of the integrin subunit
mRNA generates a variant form,
, with a unique
cytoplasmic domain that differs from
for a 48-amino
acid COOH-terminal sequence. The potential role of this unique sequence
in modulating cellular functions was investigated using Chinese hamster
ovary (CHO)
cells transiently transfected with cDNAs coding
for human integrin
or
subunits or
mutants containing truncated forms of the
cytoplasmic domain. A differential effect of
and
on cell proliferation was observed.
Expression of wild type
was associated with a
6-10-fold increase in cell proliferation in response to serum, as
measured by [
H]thymidine incorporation. In
contrast, only a 2-fold increase in cell proliferation was observed in
transfectants expressing comparable levels of
. Cells
expressing the
mutant truncated at Leu
and lacking the last 31 amino acids of the cytoplasmic domain
showed a 12-fold proliferation increase in response to serum. However,
three
deletion mutants, lacking the COOH-terminal
23, 13, and 8 amino acids, which all contained residues
Gln
-Gln
of the variant cytoplasmic
domain responded to serum stimulation with a 2-fold increase in
[
H]thymidine uptake. The effect of
expression on cell proliferation was not associated with changes
in exposure of integrin functional epitopes, as judged by the finding
that CHO transfectants expressing
, full-length or
deletion mutants, or
equally adhered to a
functionally inhibitory monoclonal antibody against human
integrin. Expression of
inversely correlated
with the mitogenic potential of vascular cells. Absent on growing
cultured endothelial cells, surface expression of
was induced in growth-arrested, tumor necrosis factor-stimulated
endothelial cells. These findings suggest that integrin alternative
splicing may provide an accessory mechanism to modulate cell
type-specific growth regulatory pathways during vascular cell injury in vivo.
The interactions of cells with extracellular matrix are predominantly regulated by the integrin family of cell surface receptors(1, 2, 3) .
Integrin functions,
such as affinity for the ligand, subcellular localization, and
signaling events, are regulated by the short cytoplasmic domain of both
and
subunits(4, 5) . The cytoplasmic
domain of
modulates
migration(6, 7) , integrin
localization(8, 9) , and focal adhesion kinase
activation (6, 10, 11, 12) . Recent
studies have also suggested that the
subfamily and
its cytoplasmic domain participate in the control of cell
differentiation (13) and proliferation. In the latter case, it
has been shown that, first,
-ligation prevents
apoptosis(14, 15) ; second,
integrins regulate cell growth and survival in normal human
breast epithelium (16) and in other cells (17) ; and
finally, domain-swapping of the integrin
subunit
cytoplasmic domain differentially regulates the effect of fibronectin
and of an integrin-activating antibody on cell
proliferation(18) .
We have previously described
(formerly
), an alternatively
spliced form of the ubiquitous integrin
(also
designated
) subunit which contains a unique 48-amino
acid sequence in its cytoplasmic domain (19) and has no
homology with
cytoplasmic domain(20) . Based
on the hypothesis that variant sequences in the
cytoplasmic domain might differentially modulate cell proliferation, we
have investigated the ability of the integrin
subunit and of
deletion mutants, truncated in
the cytoplasmic domain, to modulate cell growth.
We show here that a
unique sequence Gln-Gln
in the
cytoplasmic domain negatively regulates cell growth
and that
expression correlates with a
growth-arrested phenotype in cytokine-stimulated, vascular endothelial
cells.
Authenticity of the cDNA construct was
first established by in vitro translation and
immunoprecipitation. The translated polypeptide, which showed the
expected molecular mass of
100 kDa, was specifically
immunoprecipitated by an antiserum against the 785-808
cytoplasmic domain peptide or by P4C10, a monoclonal
antibody that recognizes the extracellular portion of human
integrin (Fig. 1A), but not by an antiserum
directed to the wild type
cytoplasmic domain (Fig. 1A). While genetically engineered cell lines
stably expressing the
integrin could not be
obtained, transient surface expression of
was
detected in CHO cells (Fig. 1B and not shown). In
contrast, both transient (Fig. 1B) and stable (data not
shown; (25) ) transfectants expressing the wild type
were obtained. Relatively low levels of
and wild type
surface expression (Fig. 1B) were reproducibly observed under these
experimental conditions. The inability of
to be
stably expressed in transfected cells prompted additional
investigations on the possible regulatory role of this integrin variant
in growth inhibition mechanisms. For these studies, CHO cells were
transiently transfected with
,
, or
deletion mutants lacking the COOH-terminal 31, 23,
13, or 8 amino acids (Table 1). Selection based on flow
cytofluorometric sorting with P4C10 antibody against the human
extracellular domain allowed us to obtain transfected
cells expressing comparable levels of all the integrin variants (Fig. 2, A and B). To further select cells
expressing the exogenous integrins, the sorted CHO cells were panned on
plates coated with 13, a functionally inhibitory monoclonal antibody to
human
(10, 25) . The attached cells
were tested in proliferation assays to compare the
,
, or
deletion mutant functional
activities on cell proliferation. Expression of
in
CHO cells caused a marked decrease of cell proliferation as measured by
[
H]thymidine uptake and as compared with the
levels of proliferation of wild type
transfectants (Fig. 3A). FACS analysis performed using monoclonal
antibody 7E2 (against hamster
) revealed comparable
levels of expression of endogenous hamster
integrin
in the
and
transfectants and in
mock-transfected CHO cells (data not shown), thus suggesting that the
observed effect of
on cell proliferation was not due
to changes in the endogenous
integrin expression
pattern. The increase of proliferation of the
transfectants varied between 6- and 10-fold (Fig. 3, A and C), and the results were not statistically different
from the values of [
H]thymidine uptake measured
using mock-transfected CHO cells (1,248 ± 207 cpm, in the
absence of FCS and 6,463 ± 245 cpm, in the presence of 10% FCS).
In agreement with our observations, stable expression of
did not affect the increase of CHO cell proliferation triggered
by 10% FCS as compared with mock-transfected cells(18) .
Expression of
802,
812,
and
817 mutants were equally effective in
inhibiting cell proliferation (Fig. 3, A and B). In contrast, CHO cells transfected with
794 mutant proliferated in response to serum as efficiently
as wild type
transfectants (12-fold increase in
[
H]thymidine uptake as compared with unstimulated
cells; Fig. 3, C and D). The
802,
812, and the
817 transfectants responded to serum stimulation, with only
a 2-fold proliferation increase (Fig. 3D).
Figure 1:
in vitro translation and transient expression. A, human
cDNA (lanes 1-5) or control
luciferase cDNA (lanes 6 and 7) were in vitro transcribed and translated. Immunoprecipitation was performed
using rabbit antisera raised against
785-808
peptide (a-
; lanes 1 and 6), against
765-798 peptide (a-
; lane 2), preimmune serum (lane 3), or monoclonal antibodies P4C10 (a-
mAb; lanes 4 and 7) or
1C10 (Control mAb; lane 5). The immunoprecipitates
were separated on 7.5% SDS-PAGE in the presence of 2-mercaptoethanol
and visualized by autoradiography. Prestained marker proteins in
kilodaltons are shown. The arrow indicates the expected
molecular mass of the
translated product. B, CHO cells were transiently transfected with
or
cDNAs or pBJ-1 and analyzed by FACS using
P4C10, monoclonal antibody to human
, followed by
fluorescein isothiocyanate-conjugated goat anti-mouse IgG. The
fluorescence intensity is expressed in arbitrary units. Dashed
line, pBJ-1; thick line,
; thin
line,
.
Figure 2:
CHO cell surface expression of
,
, and
deletion
mutants. CHO cells were transiently transfected with
,
,
794,
802,
812, or
817 cDNAs. After transfection, the cells were
starved for 48 h, stained using P4C10 monoclonal antibody to human
, followed by fluorescein isothiocyanate-conjugated
goat anti-mouse IgG, and sorted by FACS to isolate the cells expressing
comparable surface levels of
,
,
794,
802,
812, or
817. A and B show the profiles of the sorted transfectants from two separate
experiments.
Figure 3:
DNA synthesis of CHO cell transfectants
expressing ,
, and
deletion mutants. CHO cell transfectants, expressing the
full-length
or
or the truncated
forms, were isolated by flow cytometric sorting as
in Fig. 2, and then 9,000 (panel A), 12,000 (panel
B), or 6,000 (panel C) cells/well were added, in the
absence of FCS, to 96-well plates coated with antibody 13 (10
µg/ml). Attached cells were incubated for 15 h at 37 °C in the
absence or in the presence of 10% FCS and then for 3 h at 37 °C
with 1 µCi/100 µl/well [
H]thymidine to
measure their proliferative response to FCS. A and B show the mean of duplicate observations ± S.E. values of
thymidine incorporation in the absence (1, 3, and 5) or in the presence of 10% FCS (2, 4, and 6). C, data are expressed as percent of proliferation
of the
transfectants. Values indicate
[
H]thymidine uptake measurements in the absence (1 and 3) or in the presence of 10% FCS (2 and 4). Results are mean ± S.E. values of
triplicate determinations. D, data from two to three different
experiments are expressed as -fold increase of proliferation, where the
proliferation of serum-deprived cells was normalized to 1. Data are
mean ± S.E. Group differences were compared using one-way
analysis of variance followed by Bonferroni post hoc contrast.
The difference in proliferation between
794-CHO
cells and either
802-CHO,
812-CHO cells, or
817-CHO cells is
statistically significant (p = 0.003; p = 0.004; p = 0.005, respectively). No
significant differences were found comparing
[
H]thymidine uptake of
802-CHO with
812-CHO or with
817-CHO cells.
Despite
the profound effect on cell proliferation mediated by the various
integrin variants, cell adhesion of -CHO
transfectants to the functionally inhibitory antibody 13 did not differ
from that of the wild type
-CHO transfectants. In
experiments parallel to those shown in Fig. 3A, the
transfectants expressing wild type
,
, or
deletion constructs were
analyzed for their adhesive properties to antibody 13. The number of
wild type
-CHO transfectants, attached to antibody
13-coated plates (3,200 ± 712 cells/well), did not differ from
that observed using
-CHO (3,920 ± 424
cells/well) or
802-CHO transfectants (3,200
± 480 cells/well). In Fig. 3C, the attached
cells were 3,100 ± 172 cells/well for
-CHO and
4,240 ± 320 cells/well for
794-CHO
transfectants. Similarly, no differences were observed comparing the
remaining mutants (not shown). These findings suggest that expression
of the variant
cytoplasmic domain does not affect
ligand recognition and/or adhesive properties mediated by the intact
heterodimeric receptor. It is unlikely that the association of
with various
subunits plays a role in the
observed effect since previous studies have demonstrated that
cytoplasmic domains do not regulate the
pairing
specificities, which appears to be controlled by extracellular
determinants(31) .
These results demonstrate that expression
of the integrin subunit exerts a negative regulatory
function on CHO cell proliferation, and that this effect appears to be
restricted to residues 795-802 in the variant cytoplasmic domain.
Although we might speculate that the
Gln
-Gln
region may play a direct role
in the interaction with as yet unidentified intracellular ligand(s),
other mechanisms, including secondary structure and conformational
remodeling, cannot be presently excluded.
We have here described for
the first time a role for the sequence
Gln-Gln
in mediating
effect on cell proliferation. Recent studies have highlighted a
role for
in negatively regulating cell cycle
progression in late G
, when
cDNA was
microinjected in model 10T1/2 fibroblasts(32) . In that study,
using a different cell type and experimental system, a role for only
the extreme 13 carboxyl-terminal amino acids was shown in the
inhibitory effect of cell cycle progression. This may be a consequence
of the normal (33) as opposed to the tumorigenic CHO (34) cell phenotypes used in the two studies. Furthermore, in
the present study, direct ligand engagement of
integrin by the functionally blocking antibody 13 in the absence
of serum and/or matrix deposited by the cells might have unmasked a
highly selective role for the Gln
-Gln
sequence in regulating cell growth.
In order to explore a
potential pathophysiological role for in regulation
of cell growth, endothelial cells were stimulated with inflammatory
cytokines to induce a growth-arrested phenotype(35) .
Consistent with previous observations demonstrating absence of
transcript in growing endothelium(19) , an
antiserum specific for
did not immunoprecipitate any
labeled polypeptide from surface-iodinated HUVEC (Fig. 4). In
contrast, immunoprecipitation studies from TNF
-stimulated HUVEC
revealed detectable surface levels of
integrin (Fig. 4). In parallel experiments, immunohistochemical staining
with the antiserum to
characterized in Fig. 1, demonstrated prominent expression in severely injured
and growth-impaired liver tissue.
Combined with the data
presented above, we speculate that during inflammatory responses
associated with prominent cytokine release, preferential expression of
by the injured cells may contribute to a low or
non-proliferative phenotype that may impair tissue repair and
compromise its regenerative response(s).
Figure 4:
TNF -stimulated endothelial cells
express
. HUVEC were incubated for 48 h at 37 °C
in the presence of TNF
(lanes 1, 3, and 5) or in its absence (lanes 2 and 4) and
then surface-iodinated and immunoprecipitated using antibodies to
(a-
, lanes 1 and 2);
(a-
, lanes 3 and 4), and normal rabbit serum (Control
serum; lane 5). The immunoprecipitates were separated
using 7.5% SDS-PAGE in the presence of 2-mercaptoethanol and visualized
by autoradiography. Prestained marker proteins in kilodaltons are
shown.