* Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037; and Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York,
10021
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Abstract |
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Activation of integrins upon binding to extracellular matrix proteins is believed to be a crucial
step for the regulation of cell survival and proliferation.
We have used integrin 1-null mice to investigate the
role of this collagen receptor in the regulation of cell
growth and survival in vivo.
1-deficient animals, which
are viable and fertile, have a hypocellular dermis and a deficiency in dermal fibroblast proliferation as embryos. In vitro analysis of
1-null embryonic fibroblasts
has revealed that their proliferation rate is markedly reduced when plated on collagenous substrata, despite
normal attachment and spreading. Moreover, on the
same collagenous matrices,
1-null fibroblasts fail to recruit and activate the adaptor protein Shc. The failure
to activate Shc is accompanied by a downstream deficiency in recruitment of Grb2 and subsequent mitogen-activated protein kinase activation. Taken together
with the growth deficiency observed on collagens, this
finding indicates that the
1
1 is the sole collagen receptor which can activate the Shc mediated growth
pathway. Thus, integrin
1 has a unique role among the
collagen receptors in regulating both in vivo and in
vitro cell proliferation in collagenous matrices.
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Introduction |
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INTEGRINS are transmembrane heterodimeric receptors
for extracellular matrix (ECM)1 molecules (Hynes,
1992). They are composed of
and
subunits that
heterodimerize to produce >20 different receptors. One
family of integrins, which all have the
1 subunit in common, include high affinity receptors for many ECM proteins including fibronectin, laminin, and the collagens.
Three members of this family are known to be the major
receptors for collagens, namely
1
1,
2
1, and
3
1
(Yamamoto and Yamamoto, 1994
; Gardner et al., 1996
).
All three of these receptors are expressed by fibroblasts,
and they seem to regulate different functions. Integrin
1
1, for example, appears to regulate collagen synthesis,
whereas
2
1 mediates collagen gel contraction (Schiro et
al., 1991
; Langholz et al., 1995
; Broberg and Heino, 1996
).
Integrin 1
1 is widely expressed in the adult animal,
being found in visceral and some vascular smooth muscle,
liver, microvascular endothelium, activated lymphocytes,
and dermis (see references cited in Gardner et al., 1996
).
During embryonic development,
1 is first expressed in
trophoblast immediately after implantation (Sutherland
et al., 1993
), suggesting an essential role of this molecule
during early stages of development. To analyze the role of
1 integrin during mouse formation, we have generated
mice deficient in integrin
1 by gene targeting (Gardner
et al., 1996
). This disruption prevents the formation of the
1
1 receptor without altering other
1 integrin heterodimers. Animals homozygous for the disruption develop normally and are fertile, indicating that despite its
prominent expression during development,
1 integrin is
not essential for formation of the adult mouse. Using embryonic fibrobasts derived from mutant animals, we have
shown that
1 uniquely confers on fibroblasts the ability to
adhere and migrate on collagen IV, while being only one
of three integrin collagen type I receptors present on fibroblasts, together with
2
1 and
3
1. Recent studies
have also implicated DDR1 and 2, "orphan" tyrosine kinase receptors, as receptors for collagens (Shrivastava et
al., 1997
; Vogel et al., 1997).
The interaction between integrins and their ligands, besides mediating cell adhesion, plays a role in a number of
cellular processes, including cell differentiation (Adams
and Watt, 1993), cell cycle progression (Giancotti, 1997
),
and cell survival (Frisch and Ruoslahti, 1997
). The regulation of these different events requires the activation of
specific cellular targets, including protein kinase C (Clark
and Brugge, 1995
), PI3-OH kinase (Khwaja et al., 1997
),
and the small GTPase Rho (Schwartz et al., 1996
). Moreover, there is evidence that integrins activate shared as well as subgroup-specific signaling pathways that contribute to organization of the cytoskeleton and activation of
the mitogen-activated protein kinase (MAPK) cascades,
thereby influencing immediate early gene expression
(Clark and Hynes, 1997
; Giancotti, 1997
). Recent studies
have indicated that a class of integrins that includes the
1
1,
5
1,
6
4, and
v
3 receptors, is coupled to the
Ras-MAPK signaling pathway by the adaptor protein Shc
(Maniero et al., 1995
; Wary et al., 1996
; Maniero et al.,
1997
). Adhesion mediated by Shc-linked integrins promotes cell survival and progression through the G1 phase
of the cell cycle in response to mitogenic growth factors,
whereas adhesion mediated by other integrins results in
exit from the cell cycle and, in certain cases, cell death (Wary et al., 1996
; Maniero et al., 1997
). These in vitro
studies suggest that integrin
1
1 may regulate cell survival and growth in response to ECM components. We
have used integrin
1-null mice to investigate the role of
this collagen receptor in the regulation of cell survival and
proliferation in vivo. The observation that
1-null dermis
is hypocellular, prompted us to analyze fibroblasts from
control and integrin
1-deficient mice, and to compare
their growth and survival in response to ECM.
1-null fibroblasts proliferate less than their normal counterparts
both in vivo and in vitro, suggesting that survival and proliferation on collagenous substrata are events mediated
specifically and uniquely by activation of integrin
1
1.
These results suggest that
1
1 has, among many integrin
and non-integrin collagen receptors, a unique signaling function in vivo.
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Materials and Methods |
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Cell Culture and Matrix Components
Mouse embryonic fibroblasts (EFs) were isolated from wild-type and 1-null E 14.5 embryos by trypsinization (Li et al., 1992
). All cells were used
for in vitro studies at matched passage numbers 3 or 4. Cells were cultured
in DME supplemented with 2% or 10% FCS (GIBCO BRL, Gaithersburg, MD). Matrix components used in the study were bovine skin collagen I (Vitrogen 100; Collagen Corp., Palo Alto, CA), human placental
collagen IV (Sigma Chemical Co., St. Louis, MO), human fibrinogen (gift
of Z. Ruggeri, The Scripps Research Institute, La Jolla, CA), and bovine
plasma fibronectin (GIBCO BRL).
Evaluation of Fibroblast Density in the Dermis
129Sv/ter wild-type and integrin 1-null mice (6-, 12-, and 24-wk old) were
killed by cervical dislocation. The dorsal skin was shaved, removed, fixed
in formalin, and then embedded in paraffin. Sections were stained with
hematoxylin and eosin, and the number of dermal nuclei was determined
by random evaluation of six high power field views of normal, as well as
1-null dermis.
Measurement of In Vivo Proliferative Index by PCNA Labeling
Integrin 1-null and -positive control mouse embryos at day 16.5 of gestation, were obtained from timed matings. Embryos were fixed in formalin and paraffin embedded. For proliferating cell nuclear antigen (PCNA)
staining, transverse midsections of three wild-type, as well as three
1-
deficient embryos, were stained by using the mouse monoclonal IgG2a
antibody PC10 (Zymed Laboratories, Inc., South San Francisco, CA) at a
dilution of 1:200. Biotinylated secondary anti-mouse antibody and avidin/
biotin-HRP-conjugated complex were used to detect bound antibody.
Sections were then counterstained with hematoxylin and the dermal proliferative index (PCNA-positive cells/total number of counted cells × 100)
was determined by random evaluation of 12 40× field views of dermis of
each embryo. At least 900 cells around the entire circumference of the
dermis of each embryo were thus counted.
For wound healing experiments, 1-cm dorsal full thickness, cutaneous
incisions were made and immediately closed with interrupted 6/0 prolene
sutures in four wild-type and four age-matched 1-null animals. 12 d after
the incision, animals were killed and the dorsal skin was shaved, removed,
fixed in formalin, and then paraffin embedded. Sections were then subjected to PCNA staining as described above. Fibroblasts in three 40×
fields encompassing the wound were counted, and the proliferation index
expressed as number of PCNA-positive nuclei/total fibroblast number. At
least 300 cells in each wound were counted.
Cell Proliferation Assay
Control and integrin 1-null EFs were obtained from E13.5 embryos, according to the method of Li et al. (1992)
. Cells were detached from culture
dishes by trypsin, washed, resuspended in DME containing 2% or 10%
FCS, and then plated at the density of 10 × 104 cells on 24-well plates uncoated, or coated with fibrinogen (10 µg/ml), collagen I (100 µg/ml), or a
mixture of collagen I (100 µg/ml) and IV (30 µg/ml). Fibrinogen solution
was prepared in PBS, while the solutions containing collagen I were prepared in 0.1 M acetic acid, as described by Koyama et al. (1996)
. Cells were trypsinized and counted every 24 h, for a total of 72 h.
Measurement of In Vitro Proliferative Index by BrdU Labeling and FACS®
To evaluate the proliferative index, control and integrin 1-deficient EFs
were plated in presence of 2% FCS, at a density of 5 × 104 cells on 16-well
tissue culture chamber slides (Nunc, Inc., Naperville, IL) that were uncoated or coated with fibrinogen (10 µg/ml), collagen I (100 µg/ml), or a
mixture of collagens I (100 µg/ml) and IV (30 µg/ml). After 24 h, cells
were labeled with 10 µM 5'-bromo-2'-deoxy-uridine (BrdU; Sigma Chemical Co.), and incubated for a further 24 h. Cells were then washed, fixed,
and stained with anti-BrdU mAbs (Sigma Chemical Co.), followed by
biotinylated secondary anti-mouse antibody and avidin/biotin-HRP-conjugated complex (Vector Laboratories, Burlingame, CA). The BrdU labeling index (positive cells/total number of counted cells × 100) was determined by random evaluation of 10 40× fields, counting a minimum of
200 cells. For FACS® analysis, 3 × 105 serum-starved cells were plated on
6-well plates coated with fibronectin (10 µg/ml), fibrinogen (10 µg/ml) or
collagen I (100 µg/ml) in 2% FCS. After 12 h, they were trypsinized and
fixed in 70% ethanol. Cells were stained with PBS/0.1% Triton X-100/0.2 mg/ml RNase A/20 µg/ml propidium iodide, and analyzed on a Becton Dickinson FACScan® machine. S phase measurements were obtained using the Mod Fit LT v.2 program (Verity Software, Topsham, ME).
Measurement of In Vitro Apoptotic Index by DAPI Labeling
To evaluate the effect of ECM proteins on cell survival, control and integrin 1-deficient EFs were either kept in suspension, or plated in absence
of serum, at the density of 7 × 104 cells on coverslips coated with fibrinogen (10 µg/ml), collagen I (20 µg/ml), or a mixture of collagen I (20 µg/ml)
and IV (10 µg/ml). After 16 h, coverslips were washed with PBS to remove unattached cells, and then fixed and stained with 0.5 µg/ml DAPI (Sigma Chemical Co.)/PBS/0.1% Triton X-100. Chromatin condensation was used as morphological index for apoptotic cells. The apoptotic labeling index (cells with condensed chromatin/total number of counted cells × 100) was determined by random evaluation of 5-10 40× fields, counting a
minimum of 300 cells.
In Vitro Immunoprecipitation Assay
Cell lysates were prepared according to the procedure described by Wary
et al., 1996. Briefly, control and integrin
1-null EFs were growth factor
starved for 8 h, and subsequently trypsinized. Cells were then resuspended
in DME containing 0.1% BSA and kept at room temperature for 30 min.
They were then either left in suspension or plated on dishes coated with fibronectin (10 µg/ml), collagen I (20 µg/ml), or a mixture of collagen I (20 µg/ml) and IV (10 µg/ml) all diluted in PBS. After 1 h at 37°C, the cells were washed with PBS, scraped with a rubber spatula in 50 mM Hepes, pH 7.5, 150 mM NaCl, 1% Triton X-100, and then centrifuged at 14,000 rpm at 4°C for 10 min. Protein concentration was determinded using the
micro BCA assay (Pierce Chemical Co., Rockford, IL) with BSA as the
standard. Immunoprecipitation and immunoblotting were performed as
described by Maniero et al. (1995)
. Briefly, lysates containing equal
amount of proteins were immunoprecipitated with affinity-purified anti-Shc polyclonal antibody (Maniero et al., 1995
), and subjected to immunoblotting with anti-phosphotyrosine mAb RC-20H (Transduction Laboratories, Lexington, KY), anti-Grb2 polyclonal antibody (C-23; Santa Cruz
Biotechnology, Santa Cruz, CA), and anti-Shc polyclonal antibody. Nitrocellulose-bound antibodies were detected by chemiluminescence with enhanced chemiluminescence (ECL; Amersham, Life Sciences, Little Chalfont, UK). To examine Erk activity, cell lysates containing equal amount
of proteins were immunoprecipitated with anti-Erk2 polyclonal antibody
C-14 (Santa Cruz Biotechnology) and subjected to in vitro kinase assay in
50 mM Tris, pH 7.5, 10 mM MgCl2 containing 5 µCi of [
32P]ATP (4,500 Ci/mmol; ICN Biomedicals, Inc., Costa Mesa, CA) and 2.5 µg of myelin
basic protein (Sigma Chemical Co.).
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Results |
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1-deficient Dermis Is Hypocellular
Histological analysis of skin sections from wild-type and
1-deficient mice did not reveal any substantial differences in dermal morphology and thickness (Fig. 1 A).
However, we observed that the number of dermal nuclei
was reduced in the
1-null skin when compared with the
wild-type group (Fig. 1 A). To quantify this difference,
pairs of wild-type and knockout skins obtained from 6-, 12-, and 24-wk-old 129Sv/ter mice were stained with hematoxylin and eosin, and the number of dermal nuclei was
determined by random evaluation of six high power field
views of dermis. The number of dermal nuclei in
1-null
dermis was reduced at all stages examined as compared
with the wild-type dermis (Fig. 1 B). To assess whether the
difference in cell number was due to dermal fibroblasts,
rather than dermal lymphocytes, the dermis of both control and
1-deficient animals were stained with CD5, a
specific T-lymphocyte marker. No CD5 dermal staining
was observed in the dermis of wild-type or
1-null animals
(data not shown), suggesting that dermal fibroblasts rather
than lymphocytes account for the difference in cell number observed in the two groups.
|
The observation that there are fewer fibroblasts in the
dermis of 1-null animals than in the control group suggests the possibility of a deficiency in survival or proliferation of
1-null fibroblasts in the dermis.
1-null Embryonic Fibroblasts Show Marked
Reduction in Proliferation Compared with Their
Wild-Type Counterparts
To test the hypothesis that 1
1 integrin delivers a growth
signal, the proliferative index of wild-type and
1-deficient dermal fibroblasts was assessed by staining both embryonic and adult skin with antibodies to PCNA. PCNA is a
DNA polymerase
- and
-associated protein, which is
bound tightly to DNA only during S phase (Toschi and
Bravo, 1988
); in formalin-fixed, paraffin-embedded tissues
PCNA is preferentially retained in S phase cells. Whereas results from PCNA staining are not numerically comparable to other methods of estimating S phase fraction, they
do provide robust comparisons between similarly processed samples (Holt et al., 1997
). As expected, there was
no appreciable proliferation in sections of adult dermis of
either genotype (data not shown). Analysis of 16.5-d-old
embryos, however, (Fig. 2 A) revealed a marked reduction
in the proliferative index of
1-null dermis as compared
with the wild-type (Fig. 2 B).
|
To induce fibroblast proliferation in adult dermis 1-cm
full-thickness, dorsal cutaneous incisions were generated
in age-matched wild-type and 1-null animals, and allowed to heal by primary intention. Sections of 12-d-old
wounds were stained with antibodies to PCNA (Fig. 3 A).
No differences in the proliferative index of dermal fibroblasts of either genotype were evident in the wounded areas (Fig. 3 B). We also investigated the proliferative index of different embryonic, as well as adult tissues known to
express
1
1 integrin, such as liver hepatocytes (Gullberg
et al., 1990
) and smooth muscle. No appreciable differences were observed between wild-type and
1-null embryos or adults in these tissues.
|
The finding on embryonic dermis prompted us to determine whether the differences in cell survival and proliferation observed in vivo were due to the interaction of specific ECM components with the 1
1 receptor. Therefore,
we analyzed the growth profile of fibroblasts in vitro, by
using embryonic fibroblasts, derived from control and
1-null animals, plated in the presence of low or high serum
concentration on different substrata (Fig. 4, A and B).
Whereas both wild-type and
1-null EFs showed similar growth profiles on tissue culture plastic, fibrinogen, (Fig.
4, A and B) or fibronectin (not shown), on collagen substrata
1-deficient EFs showed a marked reduction in proliferation in comparison to wild-type cells. Similar deficiency in
1-null cell growth was seen when cells were
plated on collagen IV alone (not shown). As expected, the
differences in cell growth on collagen substrata were most
pronounced in growth media containing 2% serum, and were less pronounced in the presence of 10% serum (Fig. 4
B), where both growth factors and ligands for
v and
5
integrins, vitronectin and fibronectin, were more abundant. Attempts to grow EFs in defined media in the absence of serum but containing bFGF and EGF (Wary et
al., 1996
) were unsuccessful.
|
The differences in fibroblast proliferation on collagens
was also confirmed by BrdU incorporation and anti-BrdU
staining. After 48 h of culture in 2% serum, a similar proportion of wild-type and 1-deficient EFs was BrdU positive when plated on tissue culture plastic, or fibrinogen
(Fig. 5 A). In contrast, a marked reduction in BrdU incorporation was observed in
1-null EFs plated on collagen I
(Fig. 5 A), or a mixture of collagen I and IV (Fig. 5 A).
Similar results were observed when serum-starved cells
were plated on collagenous or non-collagenous substrata
in presence of 2% FCS, and then analysed by FACS® after
12 hours (Fig. 5 B).
|
While it is clear that apoptosis occurs via distinct cellular
pathways, it is a frequent observation that circumstances
preventing cell proliferation are associated with an increase in cell death. To determine whether 1-null EFs
were more susceptible to apoptosis than their wild-type
counterparts, cells were plated on different substrata, or
kept in suspension, in the absence of serum, and the apoptotic index was determined by DAPI staining after 16 h. As expected, 60-70% of cells of either genotype became
apoptotic in suspension (Fig. 6 B), while only 2% of either
genotype plated on fibrinogen became apoptotic (Fig. 6, A
and B). On collagenous matrices however (Fig. 6, A and
B),
1-null EFs showed a sevenfold increase in apoptosis
over wild-type EFs.
|
In conclusion, the in vitro results are in agreement with
the in vivo observation, and suggest that the reduction in
dermal fibroblast number observed in 1-null mice is
caused by a combination of reduced survival and reduced
proliferation.
1-null Embryonic Fibroblasts Fail to Activate the
Adaptor Protein Shc When Plated on Collagens
The differences in proliferation between 1-null and wild-type EFs on collagen substrata suggests that the interaction between integrin
1
1 and collagens is required for
the activation of a specific survival/proliferation pathway.
It has been shown (Wary et al., 1996
) that activation of
1
1 causes recruitment and tyrosine phosphorylation
of the adaptor protein Shc. This protein, via recruitment
of Grb2 and activation of the the Ras-MAP kinase pathway, cooperates with mitogens to mediate cell survival and
proliferation. Based on this observation, we investigated
the activation of Shc by wild-type and
1-deficient EFs
upon adhesion on different substrata. We have previously
shown that
1-deficient fibroblasts fail to adhere to collagen IV (Gardner et al., 1996
). Thus, to analyze the contribution of this molecule in the activation of Shc via
1 integrin,
1-deficient cells were plated on a mixture of
collagen I and IV, or on collagen I alone, and the level of
Shc activation compared with that observed in their normal counterparts. With these substrata, the extent of adhesion of wild-type and
1-null cells is indistinguishable.
Cells kept in suspension, or plated on fibronectin were
used, respectively, as negative and positive controls for activation of the Shc pathway. Cell lysates were immunoprecipitated with anti-Shc antibody and subjected to immunoblotting with anti-phosphotyrosine antibody to determine
Shc activation, with anti-Grb2 antibody to examine recruitment of Grb2, and with anti-Shc antibody to check for
equal loading. Cell lysates were also immunoprecipitated
with anti-Erk2 antibody and subjected to in vitro kinase
assay to determine MAPK activation. As expected, EFs of
both genotype failed to cause Shc phosphorylation when kept in suspension (Fig. 7 B), despite containing normal
levels of protein (Fig. 7 A). Consequently, no recruitment
of Grb 2 (Fig. 7 C), or activation of MAP kinase (Fig. 7 D)
was observed. In contrast, both cell types activated Shc
protein and its related downstream cascade, when plated
on fibronectin (Fig. 7, A-D). Striking differences in the activation of Shc, and its related downstream events, however, were observed between wild-type and
1-null EFs
after adhesion on collagen I or a mixture of collagen I and
IV.
1-null EFs entirely failed to activate Shc when plated for 1 h on substrata of collagen I or a mixture of collagen I and IV in contrast to wild-type cells (Fig. 7 B). The failure to activate Shc by
1-deficient cells was accompanied by a
downstream deficiency in recruitment of Grb 2 (Fig. 7 C)
and subsequent MAPK activation (Fig. 7 D). Taken together with the growth deficiency we have observed in
1-null EFs on collagen substrata (Fig. 4), this finding demonstrates that
1 collagen receptor has a unique and pivotal
role in regulating cell survival and proliferation on collagenous matrices.
|
![]() |
Discussion |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study we have used integrin 1-deficient mice to investigate the role of
1
1 receptor in the regulation of
dermal fibroblast growth. We provide evidence that integrin
1
1, the major collagen-binding receptor, controls
the proliferation of dermal fibroblasts both in vivo and in
vitro, and that cell survival and proliferation on collagen
substrata are mediated specifically and uniquely by integrin
1
1.
In fibroblasts integrin 1
1 regulates different functions
upon binding to collagen matrices. One of these is the regulation of collagen synthesis. We have found that the
1-null
animals lack negative feedback regulation of collagen synthesis and synthesize excess dermal collagen in vivo. This is
not apparent morphologically, as the animals also show increased synthesis of collagenases, and thus compensate excess synthesis with increased breakdown (Gardner, H., A. Broberg, A. Pozzi, J. Heino, submitted manuscript). This
dysregulation appears to cause a small reduction in tensile strength of intact skin (Davidson, J., unpublished results).
Another function of integrin 1
1, analyzed in the
present paper, is the control of cell growth and proliferation. We have shown that fibroblast growth, proliferation,
and survival are events mediated by integrin
1 activation
of the Ras-MAPK pathway, as previously suggested by the
in vitro studies of Wary et al. (1996)
. We have demonstrated that when plated on collagen matrices,
1-deficient
fibroblasts fail to activate the adaptor protein Shc, showing a reduced cell proliferation compared with their wild-type counterparts. This observation is complemented by
our observation of dermal fibroblasts in vivo. Analysis of
embryonic skin indicates that wild-type dermal fibroblasts
proliferate more than their
1-null counterparts, supporting the hypothesis that
1 integrin plays a role in controlling cell growth. This difference in cell growth was not evident in adult skin, probably because the overall rate of
proliferation in the adult dermis is negligible. Thus, our
observation that adult
1-null dermis is hypocellular as
compared with wild-type can be interpreted as the result
of a defective proliferation of
1-deficient fibroblasts during embryogenesis, although a very slowly accumulated
deficiency in proliferation in the adult
1-null dermis cannot be ruled out by our studies.
To induce fibroblast proliferation in adult dermis, we
have performed dermal wound healing experiments on
wild-type and 1-deficient animals. 12 d after the incision,
no differences in the proliferative index of dermal fibroblasts of either genotype were evident in the wounded
area. This may be explained by the fact that, during the
first stages of wound healing fibrinogen, rather than collagen, is the major ECM component to be deposited in the
wound, accompanied by local synthesis of fibronectin.
These molecules could regulate, via
v
3 and
5
1 integrins, respectively, cell growth of both wild-type and
1-null fibroblasts, reducing or eliminating the differences in
cell proliferation visible on collagen substrata. In this context, we have observed that when plated on fibronectin both wild-type and
1-deficient embryonic fibroblasts activate the Shc pathway, and show a similar growth profile.
Fibroblasts express
5
1 (Dalton et al., 1992
) and it has
been shown that activation of
5
1 leads to recruitment of
Shc (Wary et al., 1996
). Similarly, our results on the proliferative index of wild-type and
1-deficient hepatocytes
suggest the possibility that different ECM-integrin interactions could take place in these cells, and be dominant
over the lack of
1 integrin in the control of cell survival
and proliferation. In fact, given the variety of possible ligand integrin interactions, which do promote proliferation, it is striking that embryonic dermal fibroblasts have a
perceptible dependence on the
1-collagen interaction for
proliferation in vivo.
Our in vitro observation that activation of Shc on collagenous matrices occurs only in wild-type cells indicates that, although fibroblasts express three collagen receptors 1
1,
2
1, and
3
1 (Zutter and Santoro, 1990
; Gardner et al.,
1996
),
1
1 integrin is the only one of these able to deliver a
specific collagen-induced signal to the Ras-MAPK pathway,
via Shc activation. Recently, it has been observed that the
Shc phosphotyrosine binding domain can also bind to the
DDR tyrosine kinase receptor after collagen stimulation and
receptor autophosphorylation (Vogel et al., 1997), suggesting the possibility that DDR and integrin receptors may cooperate in the regulation of cell survival and proliferation. However, the observation that the MAPK pathway is not
activated upon binding of DDR to collagen (Vogel et al.,
1997), strongly supports the hypothesis that collagen-
dependent proliferation is mainly mediated via activation
of integrin
1
1.
Integrin 1
1 is only one of a larger family of integrins
able to activate Shc. This subset of receptors includes integrin
5
1, the receptor involved in the control of cell cycle
progression and proliferation in human umbilical vein endothelial cells (HUVECs) upon binding to fibronectin
(Wary et al., 1996
) and
6
4, which plays a role in regulating keratinocyte proliferation, via activation of the
same Ras-MAPK pathway (Maniero et al., 1997
). Thus,
the same adaptor molecule, activated by different integrin-ligand interaction (
1
1-collagen,
5
1-fibronectin,
6
4-laminin, and
v
3-fibrinogen) is able to regulate
cell growth of different cell types. It is interesting to note
in this context that integrin
5-null embryos, which die in
mid-gestation, show a failure of proliferation of neural
crest derivatives (Goh et al., 1997
), suggesting that these
may depend upon the
5
1-fibronectin interaction for
proliferation in a manner analagous to the
1
1-collagen
interaction in the dermis.
Whereas activation of Shc leads to cell growth and proliferation, adhesion mediated by integrins not linked to
Shc appears unable to prevent apoptotic death (Wary et al.,
1996). We have observed that
1-null cells, although able
to adhere on collagen I or a mixture of collagen I and IV
indistinguishably from their wild-type counterparts, are
more susceptible to apoptosis, indicating that
1 integrin
effects both fibroblast proliferation and survival. Shc signaling may contribute to protection from apoptosis by activating Ras and thereby PI-3 kinase, as activated forms of
Ras and PI-3 kinase protect from suspension induced apoptosis (Khwaja et al., 1997
). FAK activation by various integrins has also been implicated in cell survival (Frisch and
Ruoslahti, 1997
; Hungerford et al., 1996
), and may too act
via PI-3 kinase (Chen and Guan, 1994
).
It is striking that the adhesive function of integrin 1
1
is apparently dispensable, permitting normal morphogenesis in the null, it is its role in regulating cell proliferation
which, in its absence, gives rise to a perceptible phenotype
in vivo. The observation that only one of the three dermal
fibroblast collagen receptors specifically controls proliferation suggests that different integrin receptors for a single
ligand accomplish different and unique functions within a
single cell. Such functions may be quite distinct from, and
in some instances more important than, their adhesive
functions.
![]() |
Footnotes |
---|
Received for publication 21 April 1998 and in revised form 9 June 1998.
H. Gardner thanks J. Trotter for training in flow cytometry, and J. Leopard for histotechnology.This work was supported by National Institutes of Health (NIH) grant R29-AR4415 and the Dr. Mark Flapan Memorial Grant Award from the Scleroderma Federation/United Scleroderma Foundation to H. Gardner, and by NIH grant CA78901 to F.G. Giancotti.
![]() |
Note Added in Proof |
---|
A recent study provides further evidence that integrin-mediated Shc signaling regulates cell proliferation in vivo (Murgia, C., P. Blaikie, N. Kim, M. Dans, H.T. Petrie, and F.G. Giancotti. 1998. EMBO (Eur. Mol. Biol. Organ.) J. In press).
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Abbreviations used in this paper |
---|
BrdU, 5'-bromo-2'-deoxy-uridine; ECM, extracellular matrix; EF, embryonic fibroblasts; MAPK, mitogen-activated protein kinase; PCNA, proliferating cell nuclear antigen.
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