* Department of Cell and Structural Biology, Department of Pediatrics, and § Department of Biochemistry, University of Illinois,
Urbana, Illinois 61801; and
Department of Anatomy and Cell Biology, State University of New York Health Science Center,
Syracuse, New York 13210
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Abstract |
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We previously demonstrated contrasting
roles for integrin subunits and their cytoplasmic domains in controlling cell cycle withdrawal and the onset
of terminal differentiation (Sastry, S., M. Lakonishok,
D. Thomas, J. Muschler, and A.F. Horwitz. 1996. J. Cell
Biol. 133:169-184). Ectopic expression of the integrin
5 or
6A subunit in primary quail myoblasts either decreases or enhances the probability of cell cycle withdrawal, respectively. In this study, we addressed the
mechanisms by which changes in integrin
subunit ratios regulate this decision. Ectopic expression of truncated
5 or
6A indicate that the
5 cytoplasmic
domain is permissive for the proliferative pathway
whereas the COOH-terminal 11 amino acids of
6A cytoplasmic domain inhibit proliferation and promote differentiation. The
5 and
6A cytoplasmic domains do
not appear to initiate these signals directly, but instead regulate
1 signaling. Ectopically expressed IL2R-
5 or
IL2R-
6A have no detectable effect on the myoblast
phenotype. However, ectopic expression of the
1A integrin subunit or IL2R-
1A, autonomously inhibits differentiation and maintains a proliferative state. Perturbing
5 or
6A ratios also significantly affects
activation of
1 integrin signaling pathways. Ectopic
5
expression enhances expression and activation of paxillin as well as mitogen-activated protein (MAP) kinase
with little effect on focal adhesion kinase (FAK). In
contrast, ectopic
6A expression suppresses FAK and MAP kinase activation with a lesser effect on paxillin.
Ectopic expression of wild-type and mutant forms of
FAK, paxillin, and MAP/erk kinase (MEK) confirm
these correlations. These data demonstrate that (a) proliferative signaling (i.e., inhibition of cell cycle withdrawal and the onset of terminal differentiation) occurs
through the
1A subunit and is modulated by the
subunit cytoplasmic domains; (b) perturbing
subunit ratios alters paxillin expression and phosphorylation and FAK and MAP kinase activation; (c) quantitative changes in the level of adhesive signaling through
integrins and focal adhesion components regulate the
decision of myoblasts to withdraw from the cell cycle, in
part via MAP kinase.
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Introduction |
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CELL proliferation and differentiation are governed
by multiple stimuli including soluble growth factors, the extracellular matrix (Juliano and Haskill,
1993; Adams and Watt, 1993
; Roskelly et al., 1995
), and direct cell to cell interactions (Gumbiner, 1996
). Whereas
each of these signals uniquely regulates mitogenic responses and gene activity, the decision of a cell to proliferate, differentiate, or undergo apoptosis, for example, is an
integrated response to its adhesive and growth factor environment (Schwartz and Ingber, 1994
; Sastry and Horwitz,
1996
). While the mechanisms by which growth factors produce mitogenic responses and regulate gene expression
are becoming clearer, the pathways through which adhesive interactions modulate these responses are only beginning to emerge (reviewed in Howe et al., 1998
).
Several studies demonstrate that integrins control proliferation and differentiation in numerous cell types (Varner et al., 1995; Watt et al., 1993
). Clustering of integrins
on the cell surface with ligand-coated microbeads induces
focal adhesion-like structures that recruit numerous mitogenic signaling proteins to integrin receptors which include growth factor receptors (Plopper et al., 1995
; Miyamoto et al., 1996
), mitogen-activated protein (MAP)1
kinase, lipid second messengers, protein phosphatases,
and small GTP-binding proteins (Miyamoto et al., 1995
).
Thus integrin-associated focal adhesions serve as signaling
centers where adhesive and mitogenic pathways can integrate. Numerous physical interactions between integrins
or focal adhesion components and mitogenic signaling proteins have been demonstrated. For example, integrins
can interact with growth factor receptors through adaptor
proteins like IRS-1 (Vuori and Ruoslahti, 1994
) and shc
(Mainiero et al., 1995
; Wary et al., 1996
). Focal adhesion
kinase (FAK) can interact with PI 3-kinase (Chen and
Guan, 1994
) and with GRB2 (Schlaepfer et al., 1994
).
Through its interaction with GRB2, FAK potentially links integrin signaling to the ras/MAP kinase pathway.
Whereas these studies show a biochemical coupling between integrin and growth factor signaling pathways, the
functional significance of these interactions in the context
of the regulation of proliferation and differentiation is not
well understood. MAP kinase stands out as a key point of
convergence between integrin and growth factor pathways
(Chen et al., 1994; Zhu and Assoian, 1995
; Miyamoto et al.,
1996
; Renshaw et al., 1997
) and is required for proliferation of most cells. However, mitogenic responses can be
controlled by pathways that do not use MAP kinase (Olson et al., 1995
; Klippel et al., 1998
). In addition, MAP
kinase can modulate other integrin-dependent cell responses including motility (Klemke et al., 1997
) and integrin activation (Hughes et al., 1997
) suggesting that its activation produces pleiotropic effects. Further, the focal
adhesion proteins FAK and paxillin, which are phosphorylated in response to many soluble mitogenic stimuli (reviewed in Sastry and Horwitz, 1996
) as well as in response
to integrin engagement are likely to play an important role
in integrin-growth factor synergy. Although recent studies
indicate FAK plays a role in cell survival (Frisch et al.,
1996
; Hungerford et al., 1996
; Ilic et al., 1998
) and motility
(Ilic et al., 1995
; Cary et al., 1996
; Gilmore and Romer, 1996
), the role of FAK and paxillin, and variations in the
level of their activation, in mitogenic signaling is not well understood.
We previously reported contrasting roles for integrin subunits in proliferative signaling using myogenic differentiation as a model system (Sastry et al., 1996
). Using ectopic expression of integrins in primary quail myoblasts we
provided clear biological evidence that integrin
subunits
uniquely alter the response of myoblasts to growth factors.
We attributed this effect in part to perturbation of integrin
subunit ratios (on the order of a three- to fivefold increase in relative expression) which strikingly shifted the
probability that a myoblast would either proliferate or
withdraw from the cell cycle and initiate terminal differentiation. Ectopic expression of the
5 integrin enhanced the
mitogenic response to favor a much increased probability
of proliferation. In contrast, ectopic expression of the
6A
integrin decreased the probability of continued proliferation and promoted differentiation. In addition, we also implicated the
subunit cytoplasmic domains in controlling proliferative versus differentiative signals through integrins.
In this study, we used ectopic expression of these two subunits in primary skeletal muscle myoblasts as a convenient tool to drive either the proliferative or differentiative pathway through integrins. We used this approach to
(a) assess the relative contribution of the individual
subunit cytoplasmic domains and (b) identify intracellular targets of integrins that modulate the probability of a myoblast to proliferate or withdraw from the cell cycle and initiate terminal differentiation. First, we demonstrate that the
subunit cytoplasmic domains indirectly regulate proliferative versus differentiative signals through the
1A
cytoplasmic domain. The
5 cytoplasmic domain is permissive for proliferative signaling while a discrete region
of the
6A cytoplasmic domain promotes cell cycle withdrawal. Furthermore, the
1A cytoplasmic domain is
sufficient to initiate proliferative signals and inhibit differentiation. Second, we show that the ectopic
subunits differentially alter the expression and/or activation
of FAK, paxillin, and MAP kinase. Ectopic expression of
paxillin or CD2-FAK and their mutants recapitulate the
effects of ectopic integrins on myoblast proliferation and differentiation. The effect of ectopic
5 or
6A on proliferation and differentiation can be reversed by altering the
relative activity of MAP/erk kinase (MEK), an upstream
activator of MAP kinase. These results suggest a model in
which proliferative signaling occurs through the integrin
1A subunit which is modulated by the
subunit cytoplasmic domains. The level of signaling emanating from the
1A subunits controls the level of FAK, paxillin, or MAP
kinase activation. Thus, in addition to changes in integrin ratios, quantitative changes in the level of focal adhesion
signaling or MAP kinase activation shift the probability
that a myoblast will proliferate or differentiate.
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Materials and Methods |
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Primary Cell Culture
Primary myoblasts were isolated from pectoralis muscle of nine day Japanese quail embryos as previously described (Konigsberg, 1979). In brief,
the breast muscle was dissected from the embryo and myoblasts were dissociated from muscle tissue with 0.1% dispase (Sigma Chemical Co.) in
PBS. The cell suspension was filtered through a Sweeney filter; cells
were seeded onto gelatin-coated tissue culture plates (0.1% gelatin in
PBS). Myoblast cultures were maintained in complete myoblast medium
(DMEM [Sigma Chemical Co.] containing 15% horse serum, 5% chick
embryo extract, 1% pen/strep, and 1.25 mg/ml fungizone [GIBCO BRL]).
Myoblasts were subcultured in trypsin-EDTA (0.06% trypsin, 0.02%
EDTA) and used between passages 1 and 10.
Antibodies and Extracellular Matrix Ligands
The muscle -actinin-specific mAb, 9A2B8, was kindly provided by D. Fishman (Cornell University, New York, NY) as a hybridoma supernatant. mAb VIF4, which recognizes the human
5 integrin extracellular domain was a gift of R. Isberg (Tufts University, Boston, MA). The chicken
6-specific polyclonal antibody,
6ex (de Curtis et al., 1991), was provided
by L. Reichardt (University of California, San Francisco, CA). mAb 2B7
directed against the extracellular domain of the human
6 integrin (Shaw
et al., 1993
), was a gift of A. Mercurio (Harvard Medical School, Boston,
MA). The mAb 165 is directed against paxillin (Turner et al., 1990
). mAb
2A7 directed against FAK (Kanner et al., 1990
) and the polyclonal Ab
BC3 directed against FAK (Shaller et al., 1992) were gifts of T. Parsons
(University of Virginia, Charlottesville, VA). The anti-FAK polyclonal
antibody C-20, was purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). The anti-active MAPK polyclonal antibody, which specifically
recognizes dually phosphorylated, activated MAPK, was purchased from
Promega. The anti-MAPK mAb that recognizes erk-1, as well as the anti-phosphotyrosine antibodies, RC20H and PY20, were purchased from
Transduction Laboratories. The anti-hemagglutinin (HA) mAb, 12CA5,
was purchased from Boehringer Mannheim. The anti-human CD2 mAb,
TS2/18.1.1 was purchased from the Developmental Studies Hybridoma
Bank. TS2/16, a mAb against human
1 integrin (Hemler et al., 1984
) was
from M. Hemler (Dana-Farber Cancer Institute, Boston, MA). Antibody
against the human IL2 receptor was purchased from Boehringer Mannheim. Poly-L-lysine was purchased from Sigma Chemical Co. Fibronectin
was purified from human plasma by affinity chromatography as previously
described (Ruoslahti et al., 1982
). Laminin was isolated from murine Englebreth-Holm-Swarm sarcoma as previously described (Kleinman et al.,
1982
).
Expression Vectors
The human 5 cDNA in pRSVneo and the chicken
6 cDNA in pRSVneo
were described previously (Sastry et al., 1996
). The chicken
61044t truncation was constructed by first subcloning a 1.6-kb HindIII-SalI fragment
of the chicken
6A DNA into M13 and then introducing an in-frame BclI
site at amino acid position 1044 (de Curtis et al, 1991). Mutants were confirmed by restriction digestion of M13 clones with BclI and by single
stranded DNA sequencing using the dideoxy-chain termination method
according to the SequenaseTM protocol (United States Biochemical
Corp.). An 800-bp BstXI-SalI fragment containing the mutation was subcloned into pRSVneo
6 partially digested with SalI and completely with
BstXI. The human
6A and
6B cDNAs, in the expression plasmid pRc/
CMV (Shaw et al., 1993
), were a generous gift of A. Mercurio (Harvard
Medical School, Boston, MA). The pRSVneo-CH8
1 plasmid was constructed by subcloning a 1-kb HindIII fragment, containing the CH8
epitope tag, from the CH8
1 pBJ-1 construct received from Y. Takada
(Scripps Research Institute, La Jolla, CA) (Takada and Puzon, 1993
) into pRSVneo
1 expression vector (Reszka et al., 1992
). pRSVIL2R-
5 and
pRSVIL2R-
1A were constructed by cloning an Nhe1-Xba1 fragment from pCMVIL2R-
5cyto or pCMVIL2R-
1A plasmids received from Susan LaFlamme (Albany Medical College, Albany, NY; Tahiliani et al., 1997
) into the Xba1 site of the pRSVneo vector. Clones were screened for
orientation by restriction digests. HA-tagged rat MEK1 and HA-tagged
rat constitutively active (CA) MEK S218/220D in pCMVneo vector i.e.,
sodium-deoxycholate, sodium-pyrophosphate, sodium-orthorandate (Catling
et al., 1995
) were received from M. Weber (University of Virginia, Charlottesville, VA). The chicken paxillin cDNA, the Y118F, and the S188/
190A mutants in the pcDNA3.0neo vector (Brown et al., 1997) were received from C. Turner. CD2FAK, CD2FAK(Y397F), and CD2FAK(K454R)
in CDM8 vector (Chan et al., 1994
) were received from A. Aruffo.
Transfection and Flow Cytometry
Cells were transiently or stably transfected using a liposome-DNA solution as previously described previously (Sastry et al., 1996). In brief, replicating myoblasts (passages 1-3) were plated on 60-mm tissue culture
plates coated with 0.1% gelatin in complete myoblast medium for 16-20 h.
Cells were incubated for 8-16 h in a solution of 8 µg of plasmid DNA and
50 µg of Lipofectamine (GIBCO BRL) in complete myoblast medium.
Transfected myoblasts were either washed with DMEM, refed with myoblast medium, and analyzed for transient expression or were trypsinized
and plated into selection medium on gelatin-coated tissue culture plates
(myoblast medium containing 0.4 mg/ml G418; GIBCO BRL) for 7-12 d
and then into myoblast medium containing 0.2 mg/ml G418 (maintenance
medium). For transfections with CD2FAK constructs, in order to generate stable populations, myoblasts were cotransfected with a pRSVneo or a
pEGFP-C1neo plasmid (Clontech, Palo Alto, CA) at 1:7 ratio (neo resistance gene:CD2FAKcDNA) and selected in G418 as previously described. The chicken
6, human
6A or
6B, and
61044t transfections were selected and maintained on laminin-coated (20 µg/ml) tissue culture
plates. For coexpression of h
6A integrin and CD2-FAK cells were
cotransfected with pRc/CMVh
6A and CDM8CD2FAK vectors at a ratio
of 1:7, respectively. Transiently transfected cells were sorted by flow cytometry (see below) for h
6A expression and the positive cells were
grown in G418 containing medium. CDM8CD2FAK vector does not carry
a neo resistance gene, therefore, only cells carrying both neo resistance
(h
6A positive) and able to replicate (CD2FAK positive) will survive.
Stable populations were analyzed both for h
6 and CD2 expression as described below.
Both transiently and stably transfected (the 5 phenotype was seen in
transient as well as stable transfectants) myoblasts were analyzed for surface expression by flow cytometry as previously described (Sastry et al.,
1996
). Chicken
6A and
61044t transfected cells were stained with a
chick
6-specific polyclonal antibody,
6ex, at 20 µg/ml in blocking buffer
(Hepes-Hanks PBS-CMF with 2% BSA) and FITC-labeled goat anti-rabbit IgG (Cappel). Human
6A or B transfected myoblasts were analyzed
with the human
6-specific mAb, 2B7, at 10 µg/ml in blocking buffer. Human
5 transfected cells were stained with VIF4 mAb. Cells transfected
with CD2FAK or its mutants were stained with anti-CD2 mAb TS2/
18.1.1. Chicken
1 transfected cells were stained with TS2/16 mAb against
the human
1 epitope. IL2R-
5 or IL2R-
1A transfected cells were analyzed with an anti-IL2R antibody. The FACS profiles of the IL2R-
5 and IL2R-cyto-transfected cells were not stable, and we were unable to obtain
populations greater than 40% positive, which were used for analysis. Flow
cytometry was performed on a EPICS cell sorter (Coulter Electronics,
Inc.) equipped with Cicero software for data analysis. As shown in Fig. 1,
expression levels were assayed by fluorescence activated cell sorting.
These profiles reflect enriched surface expression levels of ectopic integrin subunits in transfected myoblasts used in experiments.
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Cell Extracts, Western Blotting, and Immunoprecipitation
For Western blotting and immunoprecipitation experiments, untransfected and transfected myoblasts were plated on FN (UT and h5 transfected cells), on LM (UT and chicken
6 and chicken
61044t or human
6A transfected cells) or on gelatin (UT, PAX, and CD2FAK transfected
cells) for 24 h in complete myoblast medium. Since myoblasts will differentiate in the absence of serum and also secrete their own matrix, we were
unable to test the effects of specific matrix ligands on the myoblast response. Therefore, all assays were conducted in the presence of serum and
results are presented for steady state conditions. Cells were washed with ice-cold PBS containing 1 mM Na-orthovanadate and lysed in ice-cold modified RIPA extraction buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 1.0% Triton X-100, 0.25% sodium-deoxycholate, 2 mM EDTA, and 2 mM EGTA) with protease inhibitors (20 mg/ml leupeptin, 0.7 mg/ml pepstatin, 1 mM phenanthroline, 2 mM phenyl-methyl-sulfonyl-chloride, and 0.05 units aprotinin) and phosphatase inhibitors (30 mM sodium-pyrophosphate, 40 mM NaF, 1 mM sodium-orthovanadate).
Protein content of the clarified lysates was determined using the Pierce
bicinchoninic acid (BCA) method with bovine serum albumin as the standard.
For phosphotyrosine Western blots, 10-15 µg of lysates were separated
on 10% SDS-PAGE gels (Laemmli, 1970) under reducing conditions and
transferred to nitrocellulose membranes (Towbin et al., 1979
). Membranes were blocked in 1% heat denatured BSA in TST buffer (10 mM
Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween-20) overnight at 4°C. Phosphotyrosine containing proteins were detected by incubating the membranes with the anti-phosphotyrosine mAb, PY20, and a secondary horse
radish peroxidase (HRP) conjugated anti-mouse antibody (Jackson ImmunoResearch Labs) or with RC20H, a directly conjugated HRP anti-phosphotyrosine Ab. Blots were visualized by chemiluminescence (Pierce
Chemical Co.). Membranes were exposed to X-ray film (Kodak, X-OMAT
AR) and developed in an automatic film processor. When indicated,
membranes were stripped in stripping buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM
-mercaptoethanol) for 30 min at 60°C and reprobed with a different antibody.
For anti-MAPK Western blots, cells were trypsinized, washed once with soybean trypsin inhibitor (0.5 mg/ml), washed twice in Puck's Saline G (GIBCO BRL) and resuspended in serum-free medium containing 2% BSA. Cells were held in suspension for 1 h prior to plating on FN or LM in complete myoblast medium for 24 h. Cell extracts were prepared in RIPA buffer as described. 5 µg of cell lysates were separated on 12% SDS-PAGE gels under reducing conditions and the proteins transferred to nitrocellulose membranes. The membranes were blocked in 3% nonfat dry milk in TST overnight at 4°C. Active MAPK was detected by an anti-active MAPK pAb (Promega). Membranes were stripped and reprobed for total MAPK with an anti-erk1 mAb (Transduction Labs) or SC-94 anti-erk1 pAb (Santa Cruz Biotechnologies).
For paxillin, FAK, CD2-FAK, and HA immunoblot analysis, 5-20 µg cell lysates were resolved on 7.5% SDS-PAGE gels under reducing conditions and proteins transferred to nitrocellulose membranes. Membranes were blocked in TST buffer containing 3% nonfat milk and the proteins were detected with 165 mAb (anti-paxillin), BC3 pAb (anti-FAK), TS2/ 18.1.1 mAb (anti-CD2), or 12CA5 mAb (anti-HA).
For FAK immunoprecipitations, 100 µg of RIPA lysate was mixed with
1 µl of anti-FAK mAb, 2A7, 50 µl of packed agarose anti-mouse beads
(blocked in 5% BSA; Sigma) in a final volume of 500 µl. The bead-antibody-antigen complex was incubated at 4°C for 2 h with continuous agitation. For paxillin immunoprecipitations, 100 µg of cell lysate and 1 µl of
anti-paxillin mAb, 165 were incubated at 4°C with continuous agitation
for 1 h. In a separate tube, 50 µl of packed protein A-agarose beads and
30 µg/ml rabbit anti-mouse IgG were incubated in lysis buffer for 1 h. The
antigen-antibody mixture was then added to rabbit anti-mouse-protein A
beads and incubated at 4°C an additional 2 h. The beads were pelleted
gently and washed twice with lysis buffer. Bound protein was released from the beads by boiling in 100 µl Laemmli sample buffer containing 5%
-mercaptoethanol for 5 min. Equal aliquots of the precipitated protein
for each antibody were loaded onto 7% SDS-PAGE gels. The FAK IP
was blotted for FAK with C-20 and for phosphoFAK with RC20H. The
paxillin IP was blotted for paxillin with the 165 mAb or for phosphopaxillin with RC20H. All immunoprecipitations and Western blots were detected by chemiluminescence.
Immunofluorescence Staining
Cells were grown on FN- or LM-coated coverslips. Immunostaining was done at room temperature. Cells were rinsed in PBS and fixed with 3% formaldehyde in PBS for 15 min then permeabilized with 0.4% Triton X-100 in PBS for 10 min, washed and blocked in 5% goat serum in PBS (BB) for 30 min. Cells were incubated with primary Ab in BB for 30 min, washed and incubated with FITC- or rhodamine-conjugated secondary Ab (Cappel) and DAPI (Sigma Chemical Co.) for additional 30 min. Coverslips were washed extensively and mounted in medium containing elvanol and p-phenylenediamine. Fluorescence was observed on a Zeiss Axioplan microscope.
Alteration of MAPK activity and differentiation
MAP kinase activity was manipulated in h6A transfected myoblasts by
coexpression of constitutively activate (CA) MEK1. Myoblasts were
cotransfected with pRc/CMVh
6A and the HA-tagged pCMVneoMEK S218/220D vectors at a ratio of 1:7, respectively. Cells were selected in
G418 and stable populations were sorted by flow cytometry for human
6A expression as described. Cell lysates were analyzed for HA expression by Western blotting as described. Stably cotransfected cells were
plated on LM-coated plates and observed for 96 h.
To alter MAP kinase activity in h5 transfected myoblasts, h
5-expressing cells were grown in the presence of the specific MEK inhibitor
PD98059 (New England Biolabs) (Alessi et al., 1995
). Transfected myoblasts were plated on FN-coated coverslips and on FN-coated TC plates.
After 8 h in complete myoblast medium, the first dose of the inhibitor was
added to the cells at 1, 10, 25, 50, or 100 µM final concentration. Cells
were grown for an additional 24 h and a second dose of the inhibitor was
added. After 24 and 48 h in presence of the inhibitor, coverslips were fixed
and immunostained for DAPI and muscle
-actinin. At the same time,
cells were extracted in RIPA buffer as described and lysates were analyzed by Western blotting for active MAPK and total erk1 expression as
described above.
Differentiation was scored using the fusion index, which is the percentage of total nuclei in myotubes as described in Sastry et al. (1996).
![]() |
Results |
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Integrin Subunit Cytoplasmic Domains Modulate
Proliferative Signals through the
1 Subunit
We recently reported a specificity for integrin subunits
and their cytoplasmic domains in controlling the proliferative to differentiative transition in primary quail myoblasts
(Sastry et al., 1996
). Ectopic expression of the human
5
integrin subunit (h
5) enhanced the fraction of myoblasts
remaining in the proliferative phase and inhibited the initiation of terminal differentiation. In contrast, ectopic
expression of the human
6A subunit of integrin (h
6A)
inhibited myoblast proliferation and promoted differentiation. These effects resulted from a three- to fivefold increased surface expression of the
5
1 or the
6A
1 integrin (and a two- to threefold increase in total
1 integrin,
see below) with little change in the relative expression of
other integrin
subunits. These findings suggested that
the
5 cytoplasmic domain promotes proliferative signals
whereas the
6A cytoplasmic domain inhibits proliferation and enhances the fraction of cells initiating terminal differentiation.
To assess the contribution of these two cytoplasmic domains, we first examined the effect of ectopic 5 and
6A
truncation mutants on myoblast proliferation and differentiation. (Fig. 2 and Table I). As we reported previously,
ectopic expression of the h
5 truncation,
5GFFKR,
which retains only the conserved GFFKR sequence, promoted proliferation and inhibited differentiation similar to
the wild-type h
5 subunit (Sastry et al., 1996
; Table I).
These findings suggest that the majority of the
5 cytoplasmic domain is not required for proliferative signals.
On the other hand, ectopic expression of an
6A truncation,
61044t, which deletes the COOH-terminal 11 amino
acid residues, restores proliferative signaling and produces
a phenotype similar to that of the ectopic
5 subunit. Myoblasts expressing
61044t remain in the proliferative phase
and do not differentiate even in high density cultures (Fig. 2). Like h
5 transfected myoblasts (Fig. 2 A), myoblasts
expressing
61044t do not express muscle
-actinin (Fig.
2), a myogenic differentiation marker, and exhibit a fusion
index of 5% (Fig. 2 B) after 72 h of culture in a rich medium. This contrasts UT controls and h
6A transfected
cells (Fig. 2) where a significant fraction of cells express
muscle
-actinin and fuse into multinucleated myotubes (Fig. 2 B). Preliminary mapping of the COOH-terminal 11 amino acids points to S1071 (in h
6A) as a key residue,
since its mutation to alanine produces a phenotype with
enhanced proliferation (data not shown). Furthermore,
the proliferation inhibiting effect of h
6 integrin is specific
for the
6A cytoplasmic domain isoform. Ectopic expression of the h
6B subunit in myoblasts promotes proliferation and inhibits differentiation (data not shown, Table I).
Consistent with these results, the
6A isoform is the predominant
6 integrin expressed in striated muscle (Hogervorst et al., 1993
; and our own unpublished observations)
and in embryonic cells of determined lineage (Cooper et al.,
1991
) whereas
6B is highly expressed in proliferating,
totipotent or undifferentiated ES cells. Taken together,
these observations suggest that the
5 and
6A cytoplasmic domains function differently: the
5 cytoplasmic domains appears permissive whereas a discrete region of the
6A cytoplasmic domain is inhibitory with respect to proliferation.
|
|
To determine whether the 5 or
6A cytoplasmic domains act directly or indirectly, we assayed the effects of
single-subunit cytoplasmic domain chimeras (LaFlamme
et al., 1992
), IL2R-
5 or IL2R-
6A, on the ability of myoblasts to proliferate or differentiate. Ectopic expression of
either IL2R-
5 or IL2R-
6A had little detectable effect
on myoblast proliferation or differentiation (Fig. 2 B). These cells behaved much like control, untransfected
(UT) myoblasts. Thus the
subunit cytoplasmic domains
do not directly initiate signals for myoblast proliferation or differentiation.
How then do these two integrin subunits regulate proliferation and differentiation? Our observation that different integrins, e.g.,
5,
6B, and
61044t, all produce a similar phenotype suggests an hypothesis in which these
subunits influence the proliferative signaling through the
1 subunit. In this view the
5 cytoplasmic domain (as well
as that of the
6B,
61044t, and perhaps others) would
permit signaling through the
1 subunit whereas the
6A cytoplasmic domain would inhibit it. Previous studies have
shown that the
subunit cytoplasmic domain can regulate
1 integrin localization to focal adhesions (Briesewitz et
al., 1993
; Ylanne et al., 1993
) and integrin activation
(O'Toole et al., 1991
); both localization and activation,
however, are mediated by the
subunit cytoplasmic domain. Furthermore, the
1 cytoplasmic domain alone,
when expressed as a single subunit chimera, IL2R-
1, can
activate intracellular signals (Akiyama et al., 1994
). To test
this hypothesis, we first determined whether overexpression of the chicken
1A subunit of integrin would increase
the fraction of proliferative myoblasts. We chose the
1A
isoform since it is predominant in replicating myoblasts
(Belkin et al., 1996
). As reported in Sastry et al. (1996)
ectopic expression of integrin
subunits also produces a
two- to threefold increase in total
1 expression with little
change in relative expression of the other endogenous
subunit levels. The increase in total
1 expression maintains myoblasts in the proliferative phase and inhibits terminal differentiation. Myoblasts with enhanced
1A expression grow to confluency but exhibit a fusion index of
only ~10% compared with 60-70% for untransfected cells
(Fig. 2 E). Thus, increased
1A expression produces a
phenotype resembling that of increased h
5 subunit expression. This finding agrees with a similar result reported
previously for the
3 integrin (Blaschuk et al., 1997
).
Next, we addressed whether the 1A cytoplasmic domain could independently affect myoblast proliferation or
differentiation through ectopic expression of the single
subunit chimera, IL2R-
1A. Previous studies show that
this chimera localizes in focal adhesions (LaFlamme et al.,
1992
) and mediates enhanced integrin signaling (Akiyama
et al., 1994
). Myoblasts expressing IL2R-
1A remain replicative and proliferate until confluent with little detectable fusion into myotubes (Fig. 2 B). Like myoblasts
expressing ectopic
5 subunit, they also only exhibit significant cell cycle withdrawal and differentiation if cultured under serum-free conditions (Sastry et al., 1996
and
data not shown). These results demonstrate that the
1A
cytoplasmic domain is sufficient to transmit proliferative signals and inhibit differentiation and thus modulate the
growth factor response. Further, ectopic expression of
IL2R-
1A can rescue the h
6A phenotype. Myoblasts
that coexpress IL2R-
1A and h
6A integrins proliferate
and differentiate like untransfected cells (data not shown;
Table I). Taken together, these findings suggest that proliferative signaling through integrins occurs via the
1 subunit and that different
subunit cytoplasmic domains can modulate these signals. The effects of ectopic integrin subunits on myoblast proliferation and differentiation are
summarized in Table I.
Ectopic Integrins Regulate FAK and Paxillin
We next sought to determine the effect of ectopic 5 or
6A expression on
1A integrin signaling pathways. Since
integrins stimulate increased tyrosine phosphorylation of
several intracellular proteins (Burridge et al., 1992
; Kornberg et al., 1992
; Bockholdt and Burridge, 1993
; Petch et al.,
1995
; Vuori and Ruoslahti, 1995
), we assayed the phosphotyrosine profile of myoblasts expressing different
ectopic
subunits. Immunoblotting with an anti-phosphotyrosine antibody shows that myoblasts ectopically expressing the h
5 integrin contain elevated tyrosine phosphorylation of proteins migrating in the molecular mass
ranges of 120-130 and 65-70 kD whereas cells transfected
with h
6A show a marked, general decrease in tyrosine phosphorylation with no additional bands when compared
with UT controls (data not shown). These observations
are consistent with the phenotypic effects of the ectopic integrins presented above; i.e., the
subunits do not initiate
separate pathways. Thus, ectopic
5 expression permits
enhanced signaling through the
1A subunit, whereas the
6A integrin suppresses these signals.
Next, we pursued the identities of the phosphoproteins
migrating at 120-130 and 65-70 kD. Focal adhesion kinase
(pp125FAK) and paxillin (pp68) are downstream targets
of integrin signaling pathways that migrate in these molecular mass ranges (Burridge et al, 1992). Therefore, we assayed the level of FAK and paxillin tyrosine phosphorylation in UT, h5, and h
6A transfected myoblasts. A
Western blot of a FAK immunoprecipitation shows that
comparable levels of FAK were precipitated (Fig. 3 A,
lanes 1-3). The level of tyrosine phosphorylated FAK decreased in h
6A (Fig. 3 A, lane 6) versus h
5 transfected
(Fig. 3 A, lane 5) or UT myoblasts (Fig. 3 A, lane 4). However, the level of FAK tyrosine phosphorylation in h
5
and UT myoblasts does not differ (Fig. 3 A, lanes 4 and 5).
Immunodepletion of FAK from the lysate, followed by
Western blotting the supernatant for phosphotyrosine reveals an additional 120-kD band in the h
5 transfected
cells that could account for the observed increase in phosphotyrosine (data not shown). Thus, whereas FAK phosphorylation decreases in myoblasts expressing ectopic
6A
integrin, it is unaffected by ectopic
5 expression.
|
To determine if paxillin phosphorylation is differentially
regulated in UT, h5, and h
6A transfected myoblasts,
paxillin was immunoprecipitated with an anti-paxillin mAb.
In contrast to observations with FAK, we observed a major difference in the level of paxillin expression between
UT and h
5 transfected myoblasts. As seen in Fig. 3 B,
paxillin is significantly upregulated in h
5 transfected myoblasts (Fig. 3 B, lane 2) when compared to UT (lane 1) or
h
6A transfected (lane 3) myoblasts. The comparable intensity of a 55-kD band in the paxillin immunoprecipitation corresponds to reduced IgG and serves as a loading
control. In addition to elevated levels of paxillin, a phosphotyrosine Western blot of the paxillin immunoprecipitation shows a concomitant increase in tyrosine phosphorylation of paxillin in h
5 transfected myoblasts (Fig. 3 B,
lane 5) compared to UT myoblasts (lane 4). Tyrosine phosphorylation of paxillin in h
6A transfected cells (lane
6) is somewhat decreased relative to untransfected cells.
The enhanced paxillin expression observed in h
5 transfected myoblasts does not arise as a direct effect of the h
5
integrin. Myoblasts expressing IL2R-
1A also show increased paxillin expression whereas myoblasts expressing
IL2R-
5 do not (data not shown). Taken together, these results indicate that enhanced paxillin expression accompanies increased
5 (or
1A) levels, whereas decreased
FAK phosphorylation coincides with increased
6A levels. These data also suggest an uncoupling of FAK and
paxillin signaling.
Paxillin and FAK Regulate the Proliferative to Differentiative Transition
The altered expression and activation of paxillin and FAK
presented above prompted us to examine the effects of ectopic paxillin or FAK expression on myoblast proliferation and differentiation. As shown in Fig. 4 A (lane 2), the
level of paxillin expression increases when compared with
controls (Fig. 4 A, lane 1) after transfection of a paxillin
cDNA. Ectopic expression of wild-type paxillin inhibits
differentiation and results in a proliferative phenotype
(Fig. 5, Table II). Myoblasts expressing ectopic paxillin proliferate until confluent but neither fuse into multinucleated myotubes (Fig. 5) nor express muscle -actinin
(Fig. 5). This phenotype is similar to that of h
5 transfected myoblasts (Fig. 2). Thus ectopic paxillin expression
alone can recapitulate the effects of the h
5 or IL2R-
1A
integrin subunits. Paxillin expression levels in control cells
do not differ in replicating myoblasts versus differentiated
cultures (data not shown).
|
|
|
Two major sites of phosphorylation in paxillin in response to adhesion to fibronectin are Y118 and S188/190
(Bellis et al., 1997). The tyrosine phosphorylation site,
Y118, is also the site phosphorylated by FAK (Bellis et al.,
1995
). Therefore, we next tested the effect of a Y118F mutation (Fig. 5) or the double mutation S188/190A on the
myoblast phenotype. When expressed in myoblasts (Fig. 4
A, lanes 3 and 4), neither of these mutants showed the enhanced proliferation seen when wild-type paxillin was expressed. Instead the paxillin mutants (Fig. 5) exhibited a
phenotype characteristic of UT myoblasts (Fig. 5, Table
II). This result suggests that these tyrosine and serine
phosphorylation sites in paxillin participate in proliferative signaling that regulates myoblast cell cycle withdrawal.
Since FAK phosphorylation decreased in parallel with
the inhibition of proliferation in myoblasts expressing
h6A integrin, we next tested the effect of an ectopic FAK
mutant, Y397F, which lacks the autophosphorylation site
and cannot bind src-family kinases (Schaller and Parsons,
1994
), on proliferation and differentiation of myoblasts.
Since ectopic expression of soluble FAK often produces short-lived or weak phenotypes (Richardson and Parsons,
1996
), we used CD2-FAK, a membrane bound, chimeric
FAK construct, which is constitutively active (Chan et al.,
1994
). Presumably, this results from increased adhesive
signaling that arises from its constitutive membrane association and consequent localization in focal adhesions.
Ectopic expression of CD2-FAKY397F, inhibits myoblast
proliferation while promoting differentiation (Fig. 5). These
cells are reminiscent of myoblasts transfected with the h
6A integrin subunit except that their proliferation is not
inhibited as completely. They also show extensive fusion
into multinucleated myotubes (Fig. 5). Interestingly, myoblasts transfected with wild-type CD2-FAK (Fig. 4 B), remain proliferative and do not initiate terminal differentiation (Fig. 5) when compared to UT controls. Fewer than 5% of CD2-FAK-expressing myoblasts fuse into multinucleated myotubes (Fig. 5) or express muscle
-actinin (Fig.
5). Using FACS analysis of propidium iodide labeled cells
to measure G1/S progression, CD2-FAK transfected myoblasts show an increased ratio of G2 to G1 cells compared
to untransfected cells (data not shown). Similarly, ectopic
expression of CD2-FAK454, which is kinase defective,
also inhibits differentiation and promotes proliferation
(Fig. 5). Ectopic expression of wild-type CD2-FAK in
h
6A transfected myoblasts results in a proliferative phenotype. Myoblasts that coexpress CD2-FAK and h
6A integrin grow to confluency and do not differentiate (Fig. 5).
In sum, the h
6A phenotype can be recapitulated by expression of a FAK mutant expressed in control myoblasts
or rescued by coexpression of an activated form of FAK.
These data suggest that one potential mechanism by which
h
6A integrin inhibits myoblast proliferation is through
altering FAK phosphorylation. Thus, changes in the level
of focal adhesion signaling, through FAK or paxillin, significantly affect the likelihood of myoblasts to proliferate
or withdraw from the cell cycle and differentiate. Table II
summarizes the effects of ectopic focal adhesion molecules.
MAP Kinase Activity Modulates Integrin-mediated Proliferation and Differentiation
Several reports implicate MAP kinase in adhesion dependent regulation of proliferation (Chen et al., 1994; Zhu
and Assoian, 1995
; Wary et al., 1996
; Miyamoto et al.,
1996
; Lin et al., 1997
). In addition, MAP kinase activation
plays an important role in muscle differentiation (Bennet
and Tonks, 1997
). Therefore, we investigated if the ectopic
integrins altered MAP kinase activation to control myoblast proliferation and differentiation. MAP kinase activation was assessed by immunoblotting cell lysates with an antibody that specifically recognizes phosphorylated, or
active, forms of the 42- and 44-kD MAP kinases. Quail
myoblasts express the 44-kD MAP kinase, erk-1, which
was detected using an anti-erk-1 mAb (data not shown).
Fig. 6 shows Western blots of active MAP kinase in myoblasts expressing ectopic integrins. In all cases, control or
transfected cells were cultured for 24 h in complete serum-containing medium before extraction. The results shown
reflect differences in steady state levels of MAP kinase activity. Compared with UT myoblasts (Fig. 6, bottom, lane
1), h
5 (lane 2), or IL2R-
1A (lane 3) transfected myoblasts contain elevated levels of active MAP kinase. In
contrast, the level of active MAP kinase in h
6A transfected cells is significantly decreased (Fig. 6, top, lane 3)
compared with controls (top, lane 1). The different intensities for control levels of active MAP kinase (top versus
bottom) is due to different exposure times of the Western
blots to film. As presented earlier, the
6A cytoplasmic
domain truncation,
61044t, did not produce the proliferation inhibiting effects of h
6A integrin, and instead promoted proliferation and inhibited cell cycle withdrawal. This also altered the level of MAP kinase activation, as assayed by Western blotting. Compared with myoblasts expressing h
6A, those expressing
61044t display enhanced
levels of active MAP kinase (Fig. 6, top, lane 2). Stripping and reprobing these membranes for total erk1 levels
showed similar expression in all cells tested (data not
shown). Therefore, the level of MAP kinase activity depends on the expression of specific integrins and their cytoplasmic domains.
|
We next investigated whether altering the activation
state of MAP kinase could reverse the h5 or h
6A induced phenotypes. We manipulated the level of active
MAP kinase through overexpression of MEK-1, an upstream activator of MAP kinase, or by addition of PD-98059, a specific MEK inhibitor (Alessi et al., 1995
).
Cotransfection of myoblasts with constitutively active
MEK (CA-MEK; Catling et al., 1995
) and h
6A integrin
restores a proliferative phenotype to myoblasts expressing
h
6A. These cells stably express both the h
6A subunit
(Fig. 7 B) and CA-MEK (Fig. 7 A) after drug selection and continue to proliferate for the lifetime of the cells in
culture. FACS analysis of propidium iodide labeled cells
shows an increased ratio of G2 to G1 cells in the h
6A/
CA-MEK cotransfectants (data not shown). As reported
previously (Sastry et al., 1996
), we were unable to isolate
cells stably overexpressing only the
6A integrin. The
h
6A-CA-MEK transfected cells are similar to the h
5
transfected myoblasts; i.e., they remain proliferative and
do not differentiate appreciably (Fig. 7 E) compared with
h
6A transfected (Fig. 7 D) or UT cells (Fig. 7 C). The
level of active MAP kinase is enhanced in h
6A-CA-MEK cells (Fig. 6, top, lane 4) when compared to h
6A (Fig. 6, top, lane 3) or UT (Fig. 6, top, lane 1) myoblasts.
Interestingly, we were unable to obtain stable expression
of CA-MEK in untransfected, control myoblasts. Presumably, excessive levels of activated MEK, or MAP kinase,
leads to increased cell death or decreased cell growth.
|
We next determined whether decreasing the level of active MAP kinase, using the specific MEK inhibitor PD-98059, would reverse the h5 phenotype. h
5 transfected
myoblasts were plated onto FN-coated plates in serum-containing medium and allowed to attach for 8-12 h. Increasing concentrations of the MEK inhibitor, PD-98059 were then added for an additional 24-48 h. With increasing inhibitor concentration, the fraction of differentiated
cells increased, whereas at high inhibitor concentrations,
the total number of cells decreased, presumably due to inhibited proliferation (Fig. 8 and data not shown). After 48 h,
h
5 transfected myoblasts treated with a 25 µM or greater
concentrations (Fig. 8 C) of the MEK inhibitor displayed
marked differentiation into myotubes compared with untreated h
5 transfected cells (Fig. 8 B) resembling UT
controls (Fig. 8 A). A Western blot for the level of active
MAP kinase shows that increasing concentrations of PD-98059 reduces MAP kinase activity in h
5 transfected cells
(Fig. 8 E). Taken together, our observations demonstrate
that quantitative changes in integrins closely parallels
changes in MAP kinase activation. Moreover, the level of
active MAP kinase appears to be a critical determinant of
myoblast proliferation versus differentiation.
|
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Discussion |
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---|
In this study we addressed the mechanisms by which integrin subunits modulate intracellular signal transduction
events that lead to phenotypic changes in cell proliferation
and differentiation. Skeletal myoblasts are well suited for
this kind of study. The probability that a myoblast withdraws from the cell cycle and initiates terminal differentiation is highly sensitive to environmental cues including
growth factors and ECM components (Sastry et al., 1996
).
The sharply contrasting effects of ectopic
5 or
6A integrin on myoblast proliferation and differentiation therefore provide a useful system to address
subunit specific
signaling and the mechanisms by which integrins regulate
the myoblast decision to withdraw from the cell cycle and
initiate terminal differentiation. The data presented here
demonstrate that the ectopic
subunits differentially regulate proliferative signaling through the
1 subunit. They
also demonstrate that paxillin, FAK, and MAP kinase serve as important regulators of cell cycle withdrawal and
the onset of terminal differentiation. Finally, our results
show that the decision to withdraw from the cell cycle and
initiate terminal differentiation is highly poised and regulated by quantitative changes in the level of MAP kinase activation, which in turn is regulated by quantitative
changes in levels of integrin signaling. These observations
provide a rationale for: (a) the apparently disparate requirements of different muscle cell lines and primary cultures for proliferation and cell cycle withdrawal, (b) the modulation of integrin subunits during muscle differentiation (Menko and Boettiger, 1987
; Muschler and Horwitz,
1991
; Bronner-Fraser et al., 1992
; Lakonishok et al., 1992
;
Blaschuk and Holland, 1994
; Boettiger et al., 1995
) and (c)
the hyperproliferative phenotypes observed when integrins are modulated in some other cell types (Carroll et al.,
1995
).
How does the 5
1 integrin potentiate signaling while
the
6
1 integrin attenuates it? Our data show that the
1A subunit (i.e., cytoplasmic domain) is sufficient to
transmit proliferative signals. The
5 subunit is permissive
and allows
1 signaling while the
6A inhibits it. Previous
studies indicate that signaling through integrins requires
receptor ligation or clustering into focal adhesions (Kornberg et al., 1991
; Miyamoto et al., 1996
). In the myoblast
system, both the ectopic
5 and
6A subunits form functional receptors with the endogenous
1 subunit for FN or LM, respectively (data not shown, and Sastry et al., 1996
).
Therefore, the differences in signaling we observe are
most likely not due to an inability to bind ligand. Furthermore, since IL2R-
1A alone stimulates proliferation, receptor-ligation does not appear necessary except perhaps
to form focal adhesions. Our observation used cells cultured on a complex ECM containing serum and secreted
FN, also indicating that receptor ligation may not be a critical factor, for the inhibitory effects of ectopic
6. Interestingly, the
5
1 and the
6
1 integrins exhibit distinct subcellular distributions in muscle when cultured on the
appropriate matrix ligand (Sastry, S., J. Muschler, and M. Lakonishok, unpublished observations). The
5
1 integrin localizes in focal adhesions on a FN substrate. In contrast, the
6
1 integrin displays a diffuse cell surface distribution on a laminin substrate. Since the initiating event for
signaling through integrins is receptor clustering, the contrasting localizations of
5
1 and
6
1 could reflect a difference in their ability to signal. The clustering of integrins
recruits signaling proteins into the focal adhesion signaling
complex (Miyamoto et al., 1995
). In this view, the
5
1 integrin (or any other
1 integrin with a permissive
subunit) recruits and activates proteins like FAK, paxillin,
and MAP kinase to stimulate signaling. The
6
1 integrin,
though able to bind to LM, is unable to recruit and/or activate the requisite signaling complex.
In the context of a model in which integrin signaling occurs primarily through the 1 subunit, how might the
subunits regulate signaling? A possible mechanism is that
the
5 and
6A cytoplasmic domains differentially regulate the accessibility of binding sites on the
1 cytoplasmic
domain. The
5 subunit (and likely several other
subunits)
would permit exposure of critical binding sites, whereas
the
6A subunit would mask them. A similar mode of regulation is proposed for the
subunit cytoplasmic domain
in the ligand-dependent localization of integrins in focal
adhesions (Briesewitz et al., 1993
; Ylanne et al., 1993
).
Other models of regulation are also possible. They include
a steric masking of the
1 cytoplasmic domain by the
6
cytoplasmic domain through interaction with an inhibitory binding protein, or through an
6-mediated signaling event that results in the activation of a phosphatase. The
observation that IL2R-
1A expression rescues the h
6A
phenotype, suggests that
6A masks some critical site on
1A cytoplasmic domain. Our observation that ectopic
IL2R-
6A does not inhibit proliferation argues against an
6
1 binding protein but does not rule it out.
The critical regulatory site(s) within the 6 cytoplasmic
domain appears to reside in the eleven COOH-terminal
amino acids of the
6A sequence (Table I). This COOH-terminal region of the
6A cytoplasmic domain contains
two putative serine phosphorylation sites (Shaw et al.,
1990
; Delwel et al., 1993
), which could potentially participate in negative regulation of signaling by the
6A integrin. Preliminary mapping of the
6A cytoplasmic domain
implicates one of these phosphorylation sites in
6A
(S1071) in inhibition of proliferation (unpublished observations). However, the physiological relevance of
6 phosphorylation in this system is not yet clear.
Our observations also identify important roles for intracellular components of integrin signaling pathways in regulating cell cycle withdrawal and the onset of terminal differentiation. FAK and paxillin, stand out as potential
integrin proximal mediators of adhesive signaling. These
proteins are phosphorylated on tyrosine in response to cell
adhesion to ECM in many cell types (Guan et al., 1991;
Burridge et al., 1992
; Hanks 1992; Kornberg et al., 1992
;
Schaller et al., 1992
; Schwartz et al., 1995
). In addition, both FAK and paxillin bind to synthetic peptides derived
from the
1A cytoplasmic domain (Schaller et al., 1995
).
However, their role in cell cycle withdrawal and the onset
of terminal differentiation is not well understood. Some
evidence implicates FAK in proliferation and cell survival.
Displacement of endogenous FAK from focal adhesions in
endothelial cells, through microinjection of the focal adhesion targeting domain, interferes with cell cycle progression (Gilmore and Romer, 1996
) resulting in apoptosis.
Similarly, microinjection of
1A peptides corresponding
to the FAK binding site, or anti-FAK antibodies induces
apoptosis in cultured fibroblasts (Hungerford et al., 1996
).
Expression of constitutively active FAK, CD2-FAK, in epithelial cells protects them from apoptosis (Frisch et al., 1996
).
Our data support a requirement for FAK activation in
basal myoblast proliferation; but it does not contribute to
the enhanced proliferation, i.e., the inhibited cell cycle
withdrawal, observed in cells expressing ectopic 5. Phosphorylation of FAK on tyrosine is not significantly altered
in myoblasts expressing ectopic
5 integrin. In most cell
types, FAK phosphorylation peaks within 1 h after cell attachment to the ECM (Guan et al., 1991
; Burridge et al.,
1992
; Hanks et al., 1992
; Schaller et al., 1992
). A transient increase in FAK phosphorylation is observed in
5 transfected myoblasts after initial attachment on a FN substrate
(data not shown). However, we do not observe a sustained
increase in FAK activation that coincides with the sustained proliferative phenotype induced by ectopic
5 integrin. Additionally, overexpression of FAK, as well as several FAK mutants and FRNK (Richardson and Parsons, 1996
), had no detectable effect on myoblast proliferation
or differentiation (unpublished observations). However,
ectopic expression of CD2-FAK, a membrane-bound, activated form of FAK, does promote proliferation and inhibit differentiation, thus resembling the effect of ectopic
5 or
1A integrin. Presumably this arises because CD2-FAK is targeted to the membrane and therefore available
to recruit additional adhesive signaling complexes. In contrast, the level of FAK phosphorylation is reduced in myoblasts expressing ectopic
6A integrin, correlating with
the increased cell cycle withdrawal. Ectopic expression
of CD2-FAKY397F, which lacks an autophosphorylation
site, acts as a dominant negative and inhibits proliferation
and promotes differentiation, thus revealing its role in
basal proliferation. This phenotype is similar to but less
dramatic than that of ectopic
6A integrin expression. It is
interesting to note that Y397 is a binding site for src family
kinases (Schaller and Parsons, 1994
). Finally, coexpression
of CD2-FAK and h
6A results in proliferation. Taken together, these observations are consistent with a role for
FAK phosphorylation in the basal level of proliferation
seen in control myoblasts and show that it lies on the pathway downstream of the
6A-mediated inhibition of the
1
integrin signaling.
In contrast to FAK, both the expression and subsequent
tyrosine phosphorylation of paxillin are significantly enhanced in 5 transfected myoblasts when compared to untransfected or
6A transfected cells. A role for paxillin in
proliferation or differentiation has not been demonstrated
previously. Paxillin is a multidomain adapter protein that
mediates numerous interactions with different signaling
proteins, including FAK (Schaller and Parsons, 1994
). However, paxillin has no known enzymatic activity. Paxillin does contain multiple LIM domains, which are
involved in protein-protein interactions and targeting
of paxillin to focal adhesions (Turner and Miller, 1994
;
Brown et al., 1996
). Finally, paxillin has been implicated in
growth factor dependent differentiation where an increase
in its tyrosine phosphorylation correlates with neuronal differentiation (Khan et al., 1995
; Leventhal and Feldman,
1996
). In myoblasts, the upregulation of paxillin in response to ectopic
5 integrin coincides with a proliferative
phenotype, which can be recapitulated by ectopic expression of paxillin. Our results with paxillin mutants implicate
both the Y118F and S188/190A phosphorylation sites as
key. Whereas the mechanism by which paxillin inhibits cell cycle withdrawal, i.e., stimulates proliferation, is not known, increased paxillin expression and phosphorylation
enhances the formation of adhesive signaling complexes as
do CD2-FAK and ectopic
5 expression (unpublished observation). In contrast to the significant effects of ectopic
5 expression on paxillin expression and phosphorylation,
ectopic
6A expression had only a very modest effect on
paxillin phosphorylation and none on expression. Coexpression of paxillin and h
6A did not relieve the
6A phenotype (data not shown). Thus the level of paxillin expression and phosphorylation appear to contribute the
myoblast decision to proliferate or differentiate. However,
it may not play a role in the inhibition of proliferation by
ectopic
6A expression. It is noteworthy that our results
also indicate an uncoupling of FAK and paxillin signaling,
since they are not activated in parallel as reported for fibroblasts (Burridge et al., 1992
).
Although our results demonstrate an inhibitory role for
the 6A integrin in signaling, this integrin is capable of activating intracellular signals in other cell systems. In macrophages, for example,
6A
1-mediated attachment to
laminin leads to increased tyrosine phosphorylation of
paxillin (Shaw et al., 1995
). In addition, antibody clustering of the
6 integrin in endothelial cells leads to a distinct
profile of tyrosine phosphorylated proteins, which do not
include FAK or paxillin (Jewell et al., 1995
).
The contrasting effects of ectopic 5 or
6A integrin expression on MAP kinase activation suggests quantitative
changes in MAP kinase activation plays a major role in
regulating myoblast cell cycle withdrawal. Perturbing the
ratio of different integrins in the cell alters the level of
MAP kinase activation. In contrast to our findings, the
6A integrin, but not the
6B subunit, activates MAP kinase in macrophages (Wei et al., 1998
). Cell type differences likely explain these conflicting observations. In the
muscle system, the effect of ectopic
5 or
6A integrins
and their mutants on MAP kinase activation parallels their
phenotypic effects on myoblast proliferation and differentiation. Likewise, perturbing the activation state of MAP
kinase, using a MEK inhibitor or CA-MEK, can overcome the integrin induced effect on both MAP kinase activity
and the myoblast phenotype. Consistent with these results,
the activation of MAP kinase is apparently an important
determinant of myogenic differentiation. Inactivation of
MAP kinase by overexpression of MAP kinase phosphatase renders C2C12 myoblasts insensitive to mitogenic
stimuli, favoring expression of muscle-specific genes like
myoD (Bennet and Tonks, 1997
). Finally, our data show
that MAP kinase activation in myoblasts requires both adhesive and growth factor signals. We do not observe active
MAP kinase in the absence of serum or on a non-specific
adhesive substrate like poly-L-lysine (unpublished observations). Taken together, these findings reveal a role for
integrins in controlling myoblast proliferation and differentiation through a pathway that regulates the level of
MAP kinase activation.
The ability of different integrins to activate MAP kinase
is proposed to occur via an association of integrins with
the adaptor protein, shc. In fibroblasts or endothelial cells,
5
1 and several other integrins interact with shc and thus
can activate MAP kinase whereas the
6 integrin cannot
bind shc or activate MAP kinase (Wary et al., 1996
). However, the interaction with shc does not require integrin cytoplasmic domains. Our data do not preclude an involvement of shc; the increase in MAP kinase activation seen in
response to the
61044t truncation may reflect a difference in the binding of shc, or an unidentified adapter or
regulatory protein.
Our observations can be summarized in a working
model for adhesive regulation of myoblast withdrawal
from the cell cycle and the onset of terminal differentiation. In this model, the myoblast decision to proliferate or
withdraw from the cell cycle is regulated, at least in part,
by the level of activated MAP kinase. This decision appears highly poised and sensitive to quantitative fluctuations in the level MAP kinase activation: increased MAP
kinase activation favoring proliferation and decreased
MAP kinase activation favoring cell cycle withdrawal.
MAP kinase activation, in turn, is regulated by the signaling emanating from both adhesive and growth factor pathways. Evidently, neither pathway alone is sufficient to sustain proliferation. The absence of either growth factor or
adhesive signaling promotes cell cycle withdrawal and the
onset of terminal differentiation, which appears to function as a default. At one level, the synergy is compatible
with an additive model since increased signaling via either
enhanced growth factor or adhesive signaling leads to a
decreased probability of cell cycle withdrawal. However,
the synergy between the growth factor and adhesive signaling systems likely has interactions that are more consistent with an additive, threshold model, in which signals are
required from both the growth factor and adhesive pathways. MAP kinase is not activated and targetted to the nucleus in cells that are either adhering to a nonspecific adhesive substrate like poly-L-lysine or that lack serum
growth factors. Recent studies point to RAF as an integration point for adhesive and growth factor signals (Chen et
al., 1996; Renshaw et al., 1997; Howe et al., 1998
).
This working model provides a rationale for diverse observations on the myoblast decision to proliferate or differentiate. Infection of myoblasts with viruses encoding src
family kinases (Alemo and Tato, 1987) or erbB (Olson,
1992), a truncated form of the EGF receptor with intrinsic
tyrosine kinase activity, or the ectopic expression of CD2-FAK or paxillin all inhibit cell cycle withdrawal and produce a proliferative phenotype similar to that of myoblasts
ectopically expressing the 5 integrin. It is likely that this
arises from increased adhesive and/or growth factor signaling resulting in increased MAP kinase activation. Similarly, the general requirement for high confluency and low serum for differentiation of diverse muscle cell lines likely also appears to reflect enhanced mitogenic signaling, i.e.,
increased MAP kinase activation, which in this case results
from immortalization or adaptation to culture. Previous
reports show alterations in the expression of integrin subunits during muscle differentiation, our results raises the
possibility that they contribute to cell cycle withdrawal
and the onset of terminal differentiation. Finally, our studies appear pertinent to keratinocyte proliferation in vivo
as well as to myoblast differentiation in vitro. Transgenic mice expressing two- to threefold increased expression of
5
1 or
2
1 integrins exhibit epidermal hyper-proliferation, perturbed keratinocyte differentiation and other features of psoriasis, a skin disease (Carroll et al., 1995
). This
epidermal hyper-proliferation is similar to our observations on myoblasts expressing ectopic
5 integrin. Our
working model provides a possible mechanism. Therefore,
it is likely that quantitative changes in mitogenic signaling
through alterations in adhesive signaling produces phenotypic alterations that operate through common mechanisms in diverse systems.
![]() |
Footnotes |
---|
Address correspondence to Alan Rick Horwitz, Department of Cell and Structural Biology, B107 Chemical and Life Sciences Laboratory, 601 S. Goodwin Ave., Urbana, IL 61801. Tel.: (214) 336-6118. Fax: (214) 244-1648. E-mail: afh{at}uiuc.edu
Received for publication 2 July 1998 and in revised form 18 February 1999.
Dr. Sastry's present address is Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, NC 27599.
Dr. Horwitz's address after 1 July 1999 will be Department of Cell Biology, Box 439, Health Sciences Center, University of Virginia, Charlottesville, VA 22908.
We thank J.T. Parsons, M. Weber, Y. Yakada, S. LaFlamme, L. Reichardt, R. Isberg, M. Hemler, D. Fishman, Y. Takada, and A. Mercurio for generous gifts of reagents. We also thank A. Richardson and J.T. Parsons for their advice and M. Berg for her comments on the manuscript.
This work was supported by National Institutes of Health grants GM23244 (A. Horwitz) and GM47607 (C. Turner). C. Turner is an Established Investigator of the American Heart Association.
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Abbreviations used in this paper |
---|
BCA, bicinchoninic acid; CA-MEK, constitutively active MEK; FAK, focal adhesion kinase; HA, hemagglutinin; MAP, mitogen-activated protein; MEK, MAP/erk kinase; UT, untransfected.
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References |
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