(The FASEB Journal. 1999;13:S35-S42.)
© 1999 FASEB
Growth factor-induced signal transduction in adherent mammalian cells is sensitive to gravity
JOHANNES BOONSTRA1
Department of Molecular Cell Biology, University of Utrecht, The Netherlands
1Correspondence: Department of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands. E-mail: J.Boonstra{at}bio.uu.nl
 |
ABSTRACT
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Epidermal growth factor (EGF) activates a well-characterized signal
transduction cascade in a wide variety of cells. This activation leads
to increased cell proliferation in most cell types. Among the early
effects evoked by EGF are receptor clustering, cell rounding, and early
gene expression. The influence of gravity on EGF-induced EGF receptor
clustering and gene expression as well as on actin polymerization and
cell rounding have been investigated in adherent A431 epithelial cells
with the use of sounding rockets to create microgravity conditions.
EGF-induced c-fos and c-jun expression
decreased in microgravity. This was caused by alteration of the EGF
receptor and protein kinase C-mediated signal transduction pathways. In
contrast, neither the binding of EGF to the receptor nor the receptor
clustering were changed under microgravity conditions. Because cell
morphology was also modulated under microgravity conditions, and the
growth factor-induced signal transduction cascades have been
demonstrated to be linked to the actin microfilament system, it is
tempting to suggest that the actin microfilament system constitutes the
gravity-sensitive cell component.—Boonstra, J. Growth
factor-induced signal transduction in adherent mammalian cells is
sensitive to gravity.
Key Words: microgravity • cell proliferation • protein kinase C • actin microfilaments • cytoskeleton
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INTRODUCTION
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A NUMBER OF STUDIES have indicated that gravity
affects mammalian cell growth and differentiation (1
,
2
). Experiments in space revealed that gravity influences
many processes in humans, such as bone development, heart function, and
temporary immunocompetence (3-6)
. These latter findings
strongly suggested that, in addition to effects of gravity at the macro
level, processes at the micro level are also impaired by gravity. These
suggestions were exemplified by the observations that in human HeLa
cells an increased gravity value resulted in an alteration of gene
expression. In addition, activation of isolated human lymphocytes was
depressed under microgravity conditions (7
,
8
). These observations suggested that the observed effects
of gravity on lymphocytes and other mammalian cells originate from
gravity-dependent modulations of the molecular signaling cascades from
plasma membrane to nucleus, which lead to the onset of gene expression.
The well-characterized cellular response of human epidermal A431
carcinoma cells to epidermal growth factor
(EGF)2to some extent resembles the cascade of molecular events involved in
the activation of human lymphocytes. This was therefore considered an
appropriate model system to study the effects of gravity on molecular
processes that are fundamental in regulation of cell function,
including lymphocyte activation and bone formation. The advantages of
the model system concern the unlimited availability of the well-known
A431 cell, the faculty to keep them in culture, the existence of many
probes and antibodies required for such research, and last but not
least a thorough knowledge of the molecular machinery involved in their
activation.
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GROWTH FACTOR-INDUCED SIGNAL TRANSDUCTION
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Polypeptide growth factors, among them EGF as one of the
best-known factors to date, play a fundamental role in regulation of
cell proliferation and differentiation. Many details of the molecular
machineries that underly growth factor action have been described in
detail as discussed in many recent review articles
(9-12)
.
One of the first identified signal transduction pathways that is
activated by the EGF receptor concerns the phosphatidylinositol
bisphosphate (PIP2) turnover catalyzed by phospholipase
C
(PLC). The products formed by PLC are two second messengers,
inositol trisphosphate (IP3) and diacylglycerol (DG). DG
operates within the cell membrane and acts as an activator of protein
kinase C (PKC) (13)
, whereas IP3 is released
into the cytoplasm and raises the intracellular Ca2+
concentration by inducing its release from intracellular stores
(14)
. Ca2+ in its turn is involved in
activation of the Ca2+ and calmodulin-dependent CAM-kinase,
a serine/threonine kinase (15)
.
Another important signal transduction pathway activated by the EGF
receptor concerns the ras pathway. Ras proteins are membrane-bound
guanine nucleotide binding proteins of low molecular weight. Binding
GTP activates ras and subsequent hydrolysis of the bound GTP to GDP
causes inactivation. GTP binding can be catalyzed by guanine nucleotide
exchange factors, whereas GTPase activity can be accelerated by GTPase
activating proteins (GAPs). The formation of the active GTP.ras form is
stimulated by activated tyrosine kinases (16
,
17
). The formation of GDP.ras is stimulated by the GAP of
ras (rasGAP) and rasGAP has been demonstrated to be associated with and
phosphorylated by the EGF receptor and other tyrosine kinases
(18
, 19
). In addition, it has been reported
that the modulation of a guanine nucleotide exchange factor might be a
target for the EGF-induced activation of ras (20
,
21
).
Ras proteins have been demonstrated to act as key regulators in
mitogenesis because these proteins act as intermediates between
receptor tyrosine kinases at the plasma membrane and an intracellular
cascade of serine/threonine kinases (22
, 23
).
Thus, for example, activated ras will cause activation of Raf-1
(24)
. Recently, however, it was shown that activation of
Raf may occur independently of ras. In this case the translocation of
Raf to the plasma membrane is sufficient for the activation. It was
suggested that Ras functions as a membrane anchor for Raf, rather than
as an activator (25)
. Raf-1 is a serine/threonine kinase
that phosphorylates and activates another serine/threonine kinase MEK
(MAP/ERK kinase), which in its turn stimulates MAP kinase
(26)
. MAP kinase acts on numerous effector molecules,
including serine/threonine kinases and transcription factors, which
altogether determine the cellular response (26
,
27
).
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EFFECT OF MICROGRAVITY ON EGF-INDUCED GENE TRANSCRIPTION
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As has been outlined above, the EGF-induced signal transduction
cascade in A431 cells is considered an attractive model to study the
possible effects of microgravity on a molecular level. Because the
EGF-induced signal transduction cascades comprise a large number of
different components, including lipids, proteins, and ions as described
briefly above, it was decided to study the effect of microgravity on
EGF-induced expression of the oncogenes c-fos and
c-jun. The expression of these genes marks the end-point in
the EGF-induced signal transduction cascade. The possible effects of
altered gravity conditions were initially studied by determining the
EGF-induced rise in c-fos mRNA levels under simulated hypogravity and
hypergravity conditions. c-Fos expression was determined by
the sensitive so-called RNAse protection assay as described in detail
previously (28)
. It was demonstrated that the EGF-induced
c-fos expression was slightly depressed under simulated
hypogravity conditions as measured in a fast-rotating clinostat,
whereas the expression was increased under hypergravity conditions as
measured in the centrifuge. Subsequently these experiments were
repeated in a sounding rocket, which allowed a microgravity
(G<10-4) period of approximately 7 min. The sounding
rockets used for our studies were launched from the Esrange base in
Kiruna, northern Sweden. Users of this sounding rocket facility were
provided with ground laboratories containing standard laboratory
equipment. The payload of the rocket constituted the experimental
modules that performed the space experiments fully automatically. The
same experiments were performed simultaneously on the ground, providing
the 1-G ground control. Microgravity was reached under free-fall
conditions approximately 1 min after launch of the rocket. Immediately
after reaching microgravity, at the onset of the experiments, samples
were taken to determine whether the high G values occurring during
launch influenced the parameters of interest. After the rocket reached
its highest point, it started to descend back to earth. The experiments
were usually ended before the rocket reached the earth atmosphere. As
stated above, all parameters were determined simultaneously under
microgravity conditions and on the ground. The first studies
demonstrated clearly that EGF-induced c-fos and
c-jun expression in microgravity was reduced. On the
other hand, the expression of the constitutively expressed
ß2-microglobulin gene, which is not modulated by EGF,
remains unaffected (29)
. Quantitative analysis shows that
c-fos and c-jun expression in microgravity is
reduced by approximately 50%, as compared to the normal gravity
control samples (29)
.
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EFFECT OF MICROGRAVITY ON EGF BINDING AND RECEPTOR ACTIVATION
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One likely explanation for the observed inhibition of gene
expression may well be that microgravity leads to decreased binding of
EGF to the cell surface-located EGF receptor. Therefore EGF binding was
studied using 125I-EGF during two sounding rocket flights.
Under normal conditions, EGF binding to mammalian cells has been
analyzed in detail by use of Scatchard analysis (30-32)
.
However, microgravity did not influence the binding properties of the
receptors (33)
. These observations were in agreement with
other investigations demonstrating that microgravity conditions did not
alter receptor ligand binding (34
, 35
).
Therefore we conclude that microgravity inhibits EGF-induced signal
transduction downstream of the initial activation, that is, the binding
of EGF to its receptor.
It has been well established that the EGF receptor tyrosine
kinase is activated by EGF binding because the receptors dimerize
(36-38)
. Usually the EGF receptor dimerization is
determined by chemically cross-linking the dimers, followed by gel
electrophoresis (38)
. However, this approach is not very
well suited for application in a sounding rocket, due to the
requirements of relatively large numbers of cells. Therefore, we used
an alternative method to this end, the so-called label fracture method
(39)
.
The label fracture method, as developed by Pinto da Silva and Kan
(40)
is a method particularly suited to visualize the
lateral distribution of plasma membrane-located proteins, provided that
the proteins can be labeled by antibodies in combination with colloidal
gold particles at their external domain. This method has been used to
establish the effect of EGF on the lateral distribution of the EGF
receptor in A431 cells (39)
and it was demonstrated that
EGF caused a significant clustering of the receptors within 5 min of
addition. More recently, this method has been used to quantify the
lateral EGF receptor distribution in relation to the receptor density
(41)
. We have applied this method to establish the effect
of microgravity on the EGF-induced receptor clustering as an indication
for EGF receptor activation. Therefore A431 cells were brought into
microgravity for 5 min by use of sounding rockets during which period
the cells were treated with EGF, as described above. The cells were
then fixed and after recovery the samples were treated for label
fracture analysis according to standard protocols (41)
.
Comparison of the receptor distributions of control and EGF-treated
cells under normal gravity and microgravity conditions demonstrated
clearly that the receptor distributions in the presence and absence of
EGF were not influenced by gravity (41)
. These
observations clearly demonstrate that neither EGF binding to the
receptor nor EGF-induced receptor redistribution, and hence receptor
activation, are influenced by microgravity conditions. Thus
microgravity influences EGF-induced signal transduction downstream of
EGF binding and EGF receptor redistribution, but upstream of early gene
expression.
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MICROGRAVITY-SENSITIVE SIGNAL TRANSDUCTION PATHWAYS
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The observations described so far indicate that specific signaling
pathways are affected by microgravity. This could mean that
microgravity affects a limited number of gravity-sensitive cellular
targets. The expression of c-fos and c-jun genes
is rapidly induced by growth factors but can also be triggered by a
variety of agents that mimic the partial activation of signal
transduction pathways but that bypass the EGF receptor. Examples of
such agents are the phorbol ester phorbol 12-myristate 13-acetate
(PMA), which activates PKC, the calcium ionophore A23187, which mimics
the EGF-induced increase in the intracellular free calcium
concentration, and forskolin, which raises the intracellular cyclic AMP
concentration.
During two sounding rocket flights, A431 cells were activated with EGF,
PMA, A23187, and forskolin, respectively. Quantitative analysis of the
c-fos and c-jun expression demonstrated that both
EGF- and PMA-induced expressions were strongly inhibited under
microgravity conditions, whereas the forskolin- and A23187-induced gene
expressions were not affected. Because EGF and PMA are known to be
activators of PKC, these data indicated that the cellular response to
PKC-mediated signal transduction is a molecular target for microgravity
(42)
. These indications are supported by the observations
that the synthesis of interleukin-2 and interleukin-1, respectively, by
a human leukemic lymphocyte line and a human leukemic monocyte line
after phorbol ester stimulation were reduced in cultures under
microgravity conditions (43)
. Others reported, however, an
enhancing effect of microgravity on PKC activity (44)
.
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EFFECT OF MICROGRAVITY ON CELL MORPHOLOGY
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The data described above indicate an important role of PKC in the
gravity sensitivity of mammalian cells and these initial studies have
been supported by other studies as well (reviewed by Claassen and
Spooner, 45). However, initial experiments performed in the clinostat
and in sounding rockets indicate that, in addition to the PKC, the
cytoskeleton may be involved in the gravity sensitivity. This
suggestion is based on the observations that EGF-induced cell rounding
was enhanced under simulated microgravity conditions in the clinostat,
whereas this process was inhibited under hypergravity conditions in the
centrifuge (46)
. Cell rounding is an actin-mediated
process (47)
and therefore these observations suggest that
the actin microfilament system may be sensitive to altered gravity
conditions.
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ROLE OF THE CYTOSKELETON IN SIGNAL TRANSDUCTION
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An important aspect of the signal transduction network concerns
the efficiency with which the various components are able to influence
each other. For example, EGF-induced c-fos expression has
been determined already within 6 min after addition of EGF to the cells
(42)
, demonstrating that EGF-induced signal transduction
is very efficient. In this respect it is tempting to suggest that the
cells contain a system that is involved in the organization of a proper
localization of the various components involved in signal transduction.
The cell system that seems to be suited for such a task is the
cytoskeleton. Binding of the signal transduction components to the
cytoskeleton would allow a very precise defined localization inside the
cell. In combination with the plasma membrane, which may function as a
target for cytoplasmic signal transduction proteins as well, the
defined localization would probably facilitate various interactions
between proteins to allow the efficient signal transduction.
With respect to the feasibility of the cytoskeleton to mediate
signal transduction inside the cell, the actin microfilament system is
the most attractive component of the cytoskeleton. Actin is a highly
conserved protein, consisting of a single chain of 375 amino acids with
a molecular mass of 42 kDa. The polymerization of actin in
vivo is controlled by a diverse family of proteins, designated the
actin binding proteins. These include G-actin binding proteins, capping
proteins, severing proteins, cross-linking proteins, and membrane
attachment proteins (48)
. Of particular interest with
respect to growth factor-induced signal transduction, is the
association of the actin microfilament system with the plasma membrane
through specific actin binding proteins. These interactions are present
in three different systems, i.e. adherens junctions, focal adhesions,
and membrane skeleton. Each of these systems represent a different
signal transduction machinery with its own specific function (for
review see Boonstra et al., 9).
In addition, another important association of the actin microfilament
system with signal transduction systems has been described. This
includes the involvement of small G-proteins, especially of the Rho
family, in the regulation of actin metabolism. The Rho family of
G-proteins consists of the Rac, Rho, and Cdc42 subfamilies
(49-52)
. The Rac subfamily includes Rac1 and Rac2, Rac1
being involved in the regulation of lamellipodia and membrane ruffling
(53)
. The Rho subfamily consists of RhoA, RhoB, and RhoC.
RhoA participates in the formation of actin stress fibers, as well as
in mediating redistribution of cytoskeletal components
(49
, 53
). The Cdc42 subfamily consists of
Cdc42Hs, G25K, and RhoG (50)
and participates in the
formation of filopodia (54)
. Addition of growth factors to
cells usually results in the formation of membrane ruffles and the
disappearance of stress fibers, ultimately resulting in cell rounding
(55
, 56
). These observations clearly
demonstrate the intimate relationship between growth factor-induced
signal transduction and regulation of the cytoskeleton.
As discussed above, it is tempting to suggest that the actin
microfilament system acts as a matrix for growth factor-induced signal
transduction. This would imply that the components involved in these
signal transduction cascades are in one way or another associated with
the microfilament system.
As early as the 1980s, evidence accumulated that growth factor
receptors, among them the EGF receptor, were associated with the
cytoskeleton. Thus it was shown by biochemical and ultrastructural
methods that 20–25% of the EGF receptor population of A431 cells was
insoluble to Triton X-100 (32
, 57
,
58
) and thus associated with the cytoskeleton. In
contrast, a mutated EGF receptor in which the cytoplasmic domain of the
receptor was deleted, did not bind to the cytoskeleton
(59)
.
Subsequently, the association between the EGF receptor and the
cytoskeleton was further analyzed in detail. Selective extraction of
A431 cells into the three major cytoskeletal fractions, i.e. the
microtubules, the microfilaments, and the intermediate filaments, and
EGF binding studies on these fractions revealed that the EGF receptors
were associated with the actin microfilament system (60)
.
Because a wide variety of actin binding proteins is known to represent
substrates of the EGF receptor, it was not clear whether the receptor
binds directly or indirectly to actin. Therefore, both the EGF receptor
and actin were purified to homogeneity and subsequent co-sedimentation
assays demonstrated unequivocally that the receptor itself is an actin
binding protein with no other proteins involved (61)
.
In addition to the EGF receptor, various components involved in
EGF-induced signal transduction also appeared to be associated with the
cytoskeleton. Thus it was shown that cytoskeletons isolated from A431
cells contained high activities of PI kinase, PIP kinase, PLC, and DG
kinase (62)
. As for the EGF receptor, these proteins were
found to be associated with the actin microfilament system. It is
interesting to note that the cytoskeleton-associated kinase activities
were significantly increased on treatment of intact cells with EGF
(62)
. Recently, it was demonstrated that EGF caused an
activation and translocation of c-Src to the cytoskeleton, depending on
the actin binding domain of the EGF receptor (63)
.
These findings indeed suggest that the actin microfilament system might
function as a matrix to align the signal transduction components. In
this respect, it is of interest to mention that the EGF-induced actin
polymerization is related to membrane ruffling (55
,
56
), and that the early signal transduction events occur
predominantly in these newly formed membrane ruffles. Thus, in an
extensive study on the localization of F-actin, EGF receptor, PLC1,
and tyrosine-phosphorylated proteins in A431 cells treated with EGF for
2 and 5 min, respectively, it was shown that immediately after the
formation of the membrane ruffles, after addition of EGF to the cells,
a strong co-localization was observed between F-actin on the one hand
and EGF receptors, PLC1, and tyrosine-phosphorylated proteins on the
other. This co-localization was most apparent after 2 min of EGF
addition. In particular, the appearance of tyrosine-phosphorylated
proteins after addition of EGF was confined to the membrane ruffles,
and these tyrosine-phosphorylated proteins appeared throughout the
cells only after longer incubation periods in the presence of EGF
(64)
. These findings indicate that the membrane ruffles
represent the signal transduction organelles of the cells after
addition of EGF, and actin plays an essential role in the establishment
of these membrane ruffles.
In view of these findings, remodeling of the actin microfilament
system may have implications for the subcellular localization and
activity of molecules involved in signal transduction
(65)
. This idea is supported by observations that
inhibitors of actin polymerization modulate signal transduction
(64-68)
. Recently, we have demonstrated that
dihydro-cytochalasin B (CB), an inhibitor of actin polymerization
(69)
, inhibits EGF-induced actin polymerization
(70)
. However, co-treatment of the cells with EGF and CB
resulted in enhanced EGF receptor tyrosine kinase activity as well as
in super-induction of the c-fos gene. These data suggest
that EGF-induced actin polymerization may be important for negative
feedback regulation of signal transduction initiated by the EGF
receptor. The phosphorylation of Thr654 residue of the EGF
receptor by PKC is one of the best-characterized negative feedback
control mechanisms for signal transduction by the EGF receptor. A
synthetic peptide corresponding to the regions flanking
Thr654 of the EGF receptor was used to analyze
EGF-stimulated PKC activity by phosphorylation. Co-treatment of cells
with CB and EGF resulted in a complete loss of EGF-induced
phosphorylation of the peptide (70)
. These observations
strongly suggest that polymerized actin is obligatory for negative
feedback regulation of the EGF receptor tyrosine kinase through the PKC
pathway.
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EFFECT OF MICROGRAVITY ON THE ACTIN MICROFILAMENT SYSTEM
|
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As described above, the observed effects of microgravity on cell
morphology suggested that the actin microfilament system was sensitive
to gravity conditions. Therefore, some preliminary experiments were
performed during two sounding rocket flights in which the cells were
treated or not with EGF according to standard protocols. After fixation
and recovery of the cells, the actin filament system was labeled
specifically with fluorescent-labeled phalloidin, a toxin known for its
high affinity for filamentous actin. Analysis of the cells by
fluorescence microscopy and by fluorometry according standard
procedures (60)
revealed clearly that the F-actin content
of the cells was increased under microgravity conditions
(33)
. Addition of EGF caused a further increase in the
amount of F-actin, but no difference in activation was observed under
normal and microgravity conditions. These preliminary observations,
however, clearly indicate that the actin microfilament system may
represent a gravity-sensitive cell component.
The question that arises is whether the microgravity-sensitive PKC
pathway is related to the microgravity-sensitive component of the
cytoskeleton. Some observations suggest that this relationship does
exist. It has been known for a long time that PKC is involved in
down-modulation of the EGF receptor (71)
and that the
actin microfilament system is required for PKC translocation to the
plasma membrane (72)
. We have demonstrated that
de-polymerization of the actin microfilaments resulted in a
hyperactivation of the EGF receptor tyrosine kinase activity, probably
due to the lack of the PKC-mediated feedback (70)
. Under
microgravity conditions we have observed an increase in F-actin content
of the cells and this increase in F-actin content may cause an
increased association of PKC to the membrane. It is of interest in this
context, that an increase in PDBu, a phorbol ester that is used to
activate PKC, binding was observed in human leukocytes under
microgravity conditions (35)
, as well as an increase in
other PKC-mediated processes (44)
. Thus, the increased
level of membrane-associated PKC may cause an increase in
down-regulation of EGF receptor tyrosine kinase activity as has been
observed under microgravity conditions. This hypothesis explains the
observations that EGF-induced signal transduction is decreased under
microgravity conditions at a level downstream of receptor organization.
However, in this concept the reduction of c-fos expression
under microgravity conditions in the presence of phorbol esters is
rather puzzling. Higher PDBu binding, an increased down-modulation of
EGF receptor activity, and an increased binding of PKC to the membrane
skeleton suggests an increase rather than a decrease in PKC activity.
It should be realized, however, that the signal transduction cascade
from PKC leading to c-fos expression is not fully
understood. It could well be that specific isoforms of PKC are involved
in this signal transduction pathway and that these isoforms are
different from the ones leading to EGF receptor down-modulation. And,
as suggested recently, microgravity may influence the intracellular
distribution of the various isoforms of PKC differently
(35)
.
These findings provide, however, no clue as to the nature of the
gravity-sensitive component of the cells. Most observations seem to
indicate that the actin microfilament system represents the
gravity-sensitive component because the actin microfilament system is
modulated as soon as cells are exposed to microgravity conditions (see
above). The actin microfilament system is, however, a rather
complicated system in which many cellular proteins are involved, such
as actin-binding proteins, capping proteins, severing proteins,
cross-linking proteins, and many others (53)
. In addition,
a wide variety of cell signaling proteins and molecules are associated
with the actin microfilament system or have a role in actin
microfilament dynamics, such as the proteins of the Rho family
(49
, 50
, 54
), the molecules and
proteins involved in phosphatidylinositol metabolism
(73-75)
, and many others (60-64)
. This
complexity makes it very difficult to identify the real
gravity-sensitive component of the cells. However, in addition to a
chemical role in cellular functioning, the actin microfilament system
also has a physical role and it is therefore tempting to suggest that a
physical force, such as gravity, will have a significant effect
on the actin microfilament system. Recent physical studies of actin
networks in vitro have influenced our understanding of
cytoplasmic sol-gel transitions (76
, 77
). The
degree of entanglement, filament stiffness, and the capacity of
microgel formation within actin gels may be governed by specifically
actin-binding proteins. Therefore physical stress/response coupling may
be an important parameter during cytoskeleton-mediated signal
transduction, especially under microgravity conditions. At this time it
is still unclear whether the possible effects of gravity on the
microfilament system are due to effects on actin
polymerization/depolymerization directly or to effects on the actin
binding proteins or the Rho family members involved in the actin
metabolism. Involvement of the latter group of proteins seems unlikely,
however, because thus far no indications to this end have been
obtained. In this respect it seems attractive to study actin
microfilament systems in vitro under microgravity conditions
to obtain a better understanding of the gravity effects on cells
in vivo.
|
ACKNOWLEDGMENTS
|
|---|
I would like to thank my colleagues, Philip Rijken, Rolf de
Groot, Jeroen den Hertog, André van Puijenbroek, Arie Verkleij,
Paul van der Saag, and Siegfried de Laat for their contributions. I
would also like to thank all the other people involved in the
successful flights of the Maser 3, Maser 4, Maser 5, and Maser 6, and
SRON for financial support.
|
FOOTNOTES
|
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2 Abbreviations: EGF, epidermal growth
factor; PIP2, phosphatidylinositol bisphosphate; PLC,
phospholipase C; IP3, inositol trisphosphate; DG,
diacylglycerol; PKC, protein kinase C; GAPs, GTPase activating
proteins; PMA, phorbol myristate acetate; CB, cytochalasin B.
|
REFERENCES
|
|---|
-
Gmünder, F. K., Cogoli, A. (1988) Cultivation of single cells in space. Appl. Microgravity Tech. 9,115-122
-
Rijken, P. J., Boonstra, J., Verkleij, A. J., de Laat, S. W. (1994) Effects of gravity on the cellular response to epidermal growth factor. Adv. Space Biol. Med. 4,159-188[Medline]
-
Morey, E. R., Baylink, D. J. (1978) Inhibition of bone formation during space flight. Science 201,1138-1141[Abstract/Free Full Text]
-
Bikle, D. D., Halloran, B. P., Morey-Holton, E. (1997) Space flight and the skeleton: lessons for the earthbound. Endocrinologist 7,10-22[Medline]
-
Convertino, V. A., Doerr, D. F., Mathes, K. L., Stein, S. L., Buchanan, P. (1989) Changes in volume, muscle compartment, and compliance of the lower extremities in man following 30 days of exposure to simulated microgravity. Aviat. Space Environ. Med. 60,653-658[Medline]
-
Bungo, M. W., Charles, J. B., Johnson, P. C. (1985) Cardiovascular deconditioning during space flight and the use of saline as a countermeasure. Aviat. Space Environ. Med. 56,985-990[Medline]
-
Cogoli, A., Tschopp, A., Fuchs-Bislin, P. (1984) Cell sensitivity to gravity. Science 225,228-230[Abstract/Free Full Text]
-
Pippia, P., Sciola, L., Cogoli-Greuter, M., Meloni, M. A., Spano, A., Cogoli, A. (1996) Activation signals of T lymphocytes in microgravity. J. Biotechnol. 47,215-222[Medline]
-
Boonstra, J., Rijken, P. J., Humbel, B., Cremers, F., Verkleij, A., van Bergen en Henegouwen, P. (1995) The epidermal growth factor. Cell Biol. Int. 19,413-430[Medline]
-
Marais, R., Marchall, C. J. (1996) Control of the ERK MAP kinase cascade by Ras and Rab. Cancer Surveys 27,101-125[Medline]
-
Massagué, J., Weis-Garcia, F. (1996) Serine/threonine kinase receptors: mediators of transforming growth factor beta family signals. Cancer Surveys 27,41-64[Medline]
-
Heldin, C. H. (1996) Protein tyrosine kinase receptors. Cancer Surveys 27,7-24[Medline]
-
Kikkawa, U., Nishizuka, Y. (1986) The role of PKC in transmembrane signalling. Annu. Rev. Cell Biol. 2,149-178
-
Berridge, M. J., Irvine, R. F. (1984) Inositol triphosphate, a novel second messenger in cellular signal transduction. Nature (London) 312,315-321[Medline]
-
Hanson, P. I., Schulman, H. (1992) Neuronal Ca2+/calmodulin-dependent protein kinases. Annu. Rev. Biochem. 61,559-601[Medline]
-
Gibbs, J. B., Marshall, M. S., Scolnick, E. M., Dixon, R. A. F., Vogel, U. S. (1990) Modulation of guanine nucleotides bound to ras in NIH3T3 cells by oncogenes, growth factors, and the GTPase activating protein (GAP). J. Biol. Chem. 265,20437-20442[Abstract/Free Full Text]
-
Satoh, T., Endo, M., Nakafuku, M., Nakamura, S., Kaziro, Y. (1990) Accumulation of p21ras. GTP in response to stimulation with EGF and oncogene products with tyrosine kinase activity. Proc. Natl. Acad. Sci. USA 87,7926-7929[Abstract/Free Full Text]
-
Moran, M. F., Koch, C. A., Anderson, D., Ellis, C., England, L., Martin, G. S., Pawson, T. (1990) Src homology region 2 domains direct protein-protein interactions in signal transduction. Proc. Natl. Acad. Sci. USA 87,8622-8626[Abstract/Free Full Text]
-
Liu, X., Pawson, T. (1991) The epidermal growth factor receptor phosphorylates GTPase activating protein (GAP) at tyr-460, adjacent to the GAP SH2 domain. Mol. Cell. Biol. 11,2511-2516[Abstract/Free Full Text]
-
Buday, L., Downward, J. (1993) Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adaptor protein, and Sos nucleotide exchange factor. Cell 73,611-620[Medline]
-
Medema, R. H., de Vries-Smits, A. M. M., van der Zon, G. C. M., Maassen, J. A., Bos, J. L. (1993) Ras activation by insulin and epidermal growth factor through enhanced exchange of guanine nucleotides on p21ras. Mol. Cell. Biol. 13,155-162[Abstract/Free Full Text]
-
Bokoch, G. M., Der, C. J. (1993) Emerging concepts in the Ras superfamily of GTP-binding proteins. FASEB J 7,750-759[Abstract]
-
Khosravi-Far, R., Der, C. J. (1994) The ras signal transduction pathway. Cancer Metast. Rev. 13,67-89[Medline]
-
Zhang, X-F., Settleman, J., Kyriakis, J. M., Takeuchi-Suzuki, E., Ellidge, S. J., Marshall, M. S., Buder, J. T., Rapp, U. R., Avruch, J. (1993) Normal and oncogenic p21ras proteins bind to the amino-terminal regulating domain of c-Raf-1. Nature (London) 364,308-313[Medline]
-
Leevers, S. J., Paterson, H. F., Marshall, C. J. (1994) Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature (London) 369,411-414[Medline]
-
Daum, G., Eisenmann-Tappe, I., Fries, H-W., Troppmair, J., Rapp, U. R. (1994) The ins and outs of Raf kinases. Trends Biol. Sci. 19,474-480
-
Davis, R. J. (1994) MAPKs: new JNK expands the group. Trends Biol. Sci. 19,470-473
-
de Groot, R. P., Rijken, P. J., Boonstra, J., Verkleij, A. J., de Laat, S. W., Kruijer, W. (1991) Epidermal growth factor induced expression of c-fos is influenced by altered gravity conditions. Aviat. Space Env. Med. 62,37-40[Medline]
-
de Groot, R. P., Rijken, P. J., den Hertog, J., Boonstra, J., Verkleij, A. J., de Laat, S. W., Kruijer, W. (1990) Microgravity decreases c-fos induction and SRE activity. J. Cell Sci. 97,33-38[Abstract/Free Full Text]
-
Defize, L. H. K., Boonstra, J., Meisenhelder, J., Kruijer, W., Tertoolen, L. G. J., Tilly, B. C., Hunter, T., van Bergen en Henegouwen, P. M. P., Moolenaar, W. H., de Laat, S. W. (1989) Signal transduction by epidermal growth factor occurs through the subclass of high affinity receptors. J. Cell Biol. 109,2495-2507[Abstract/Free Full Text]
-
Boonstra, J., Mummery, C. L., van der Saag, P. T., de Laat, S. W. (1985) Two receptor classes for epidermal growth factor on Pheochromocytoma cells, distinguishable by temperature, lectins and tumor promoters. J. Cell. Physiol. 123,347-352[Medline]
-
Wiegant, F. A. C., Blok, F. J., Defize, L. H. K., Linnemans, W. A. M., Verkleij, A. J., Boonstra, J. (1986) Epidermal growth factor receptors associated to cytoskeletal elements of epidermoid carcinoma (A431) cells. J. Cell Biol. 103,87-94[Abstract/Free Full Text]
-
Boonstra, J., Rijken, P. J., de Groot, R. P., Verkleij, A. J., van der Saag, P. T., de Laat, S. W. (1997) Growth factor-induced signal transduction in mammalian cells is sensitive to gravity. Sato, A. eds. Frontiers in Biological Science in Space ,2-18 Taiyo Printing Tokyo.
-
Spooner, B. S., Guikema, J. A., Barnes, G. (1990) Binding of alpha-phetoprotein by immobilised monoclonal antibodies during episodes of zero-gravity obtained by parabolic flight. Aviat. Space Environ. Med. 61,725-728[Medline]
-
Schmitt, D. A., Hatton, J. P., Emond, C., Chaput, D., Paris, H., Levade, T., Cazenave, J-P., Schaffar, L. (1996) The distribution of protein kinase C in human leukocytes is altered in microgravity. FASEB J 10,1627-1634[Abstract]
-
Schlessinger, J. (1988) Signal transduction by allosteric receptor oligomerization. Trends Biochem. Sci. 13,443-447[Medline]
-
Cochet, C., Kashles, O., Chambaz, E. M., Borello, I., King, C. R., Schlessinger, J. (1988) Demonstration of EGF-induced receptor dimerization in living cells using a chemical covalent cross-linking agent. J. Biol. Chem. 263,3290-3295[Abstract/Free Full Text]
-
Spaargaren, M., Defize, L. H. K., Boonstra, J., de Laat, S. W. (1991) Antibody-induced dimerization activates the epidermal growth factor receptor tyrosine kinase. J. Biol. Chem. 266,1733-1739[Abstract/Free Full Text]
-
van Belzen, N., Rijken, P. J., Hage, W. J., de Laat, S. W., Verkleij, A. J., Boonstra, J. (1988) Direct visualization and quantitative analysis of epidermal growth factor induced receptor clustering. J. Cell. Physiol. 134,413-420[Medline]
-
Pinto da Silva, P., Kan, F. W. K. (1984) Label-fracture: a method for high resolution labeling of cell surfaces. J. Cell Biol. 99,1156-1161[Abstract/Free Full Text]
-
Rijken, P. J., de Groot, R. P., van Belzen, N., de Laat, S. W., Boonstra, J., Verkleij, A. J. (1993) Inhibition of EGF-induced signal transduction by microgravity is independent of EGF receptor redistribution in the plasma membrane of human A431 cells. Exp. Cell Res. 201,373-377
-
de Groot, R. P., Rijken, P. J., den Hertog, J., Boonstra, J., Verkleij, A. J., de Laat, S. W., Kruijer, W. (1991) Nuclear responses to protein kinase C signal transduction are sensitive to gravity changes. Exp. Cell Res. 197,87-90[Medline]
-
Limouse, M., Manie, S., Konstantinova, I., Ferrua, R., Schaffar, L. (1991) Inhibition of phorbol ester-induced cell activation in microgravity. Exp. Cell Res. 197,82-86[Medline]
-
Woods, K. M., Chapes, S. K. (1994) Abrogation of TNF-mediated cytotoxicity by space flight involves protein kinase C. Exp. Cell Res. 211,171-174[Medline]
-
Claassen, D. E., Spooner, B. S. (1994) Impact of altered gravity on aspects of cell biology. Int. Rev. Cytol. 156,301-373[Medline]
-
Rijken, P. J., de Groot, R. P., Briegleb, W., Kruijer, W., Verkleij, A. J., Boonstra, J., de Laat, S. W. (1991) Epidermal growth factor-induced cell rounding is sensitive to simulated microgravity. Aviat. Space Env. Med. 62,32-36[Medline]
-
Chinkers, M., McKanna, J. A., Cohen, S. (1979) Rapid induction of morphological changes in human carcinoma A431 cells by epidermal growth factor. J. Cell. Biol. 83,260-265[Abstract/Free Full Text]
-
Pollard, T. D., Cooper, J. A. (1986) Actin and actin binding proteins. A critical evaluation of mechanisms and functions. Annu. Rev. Biochem. 55,987-1035[Medline]
-
Lamarche, N., Hall, A. (1994) GAPs for Rho related GTPases. Trends Genet 10,436-440[Medline]
-
Hall, A. (1994) Small GTP binding proteins and the regulation of the actin cytoskeleton. Annu. Rev. Cell Biol. 10,31-54
-
Gutkind, J. S., Vitale-Cross, L. (1996) The pathway linking small GTP-binding proteins of the Rho family to cytoskeletal components and novel signaling kinase cascades. Cell Dev. Biol. 7,683-690
-
Lim, L., Hall, C., Monfries, C. (1996) Regulation of actin cytoskeleton by Rho-family GTPase and their associated proteins. Cell Dev. Biol. 7,699-706
-
Ridley, A., Hall, A. (1992) The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70,389-399[Medline]
-
Kozma, R., Ahmed, S., Best, A., Lim, L. (1995) The ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in swiss 3T3 fibroblasts. Mol. Cell. Biol. 15,1942-1952[Abstract]
-
Rijken, P. J., Hage, W. J., van Bergen en Henegouwen, P. M. P., Verkleij, A. J., Boonstra, J. (1991) Epidermal growth factor induces rapid reorganization of the actin microfilament system in human A431 cells. J. Cell. Sci. 100,491-499[Abstract/Free Full Text]
-
Rijken, P. J., Post, S. M., Hage, W. J., van Bergen en Henegouwen, P. M. P., Verkleij, A. J., Boonstra, J. (1995) Actin polymerization localizes to the activated epidermal growth factor receptor in the plasma membrane, independent of the cytosolic free calcium transient. Exp. Cell Res. 218,223-232[Medline]
-
Landreth, G. E., Williams, L. K., Rieser, G. D. (1985) Association of epidermal growth factor receptor kinase with the detergent-insoluble cytoskelton of A431 cells. J. Cell. Biol. 101,1341-1350[Abstract/Free Full Text]
-
Roy, L. M., Gittinger, C. K., Landreth, G. E. (1989) Characterization of the epidermal growth factor receptor associated with cytoskeletons of A431 cells. J. Cell Physiol. 140,295-304[Medline]
-
van Belzen, N., Spaargaren, M., Verkleij, A. J., Boonstra, J. (1990) Interaction of the epidermal growth factor receptor with the cytoskeleton is related to receptor clustering. J. Cell Physiol. 145,365-375[Medline]
-
van Bergen en Henegouwen, P. M. P., den Hartigh, J. C., Romeijn, P., Verkleij, A. J., Boonstra, J. (1992) The epidermal growth factor is associated with actin filaments. Exp. Cell. Res. 199,90-97[Medline]
-
den Hartigh, J. C., van Bergen en Henegouwen, P. M. P., Verkleij, A. J., Boonstra, J. (1992) The EGF receptor is an actin-binding protein. J. Cell Biol. 119,349-355[Abstract/Free Full Text]
-
Payrastre, B., van Bergen en Henegouwen, P. M. P., Breton, M., den Hartigh, J. C., Plantavid, M., Verkleij, A. J., Boonstra, J. (1991) Phosphoinositide kinase, diacylglycerol kinase, and phospholipase C activities associated to the cytoskeleton: effect of epidermal growth factor. J. Cell Biol. 115,121-128[Abstract/Free Full Text]
-
van der Heyden, M. A. G., Oude Weernink, P. A., van Oirschot, B. A., van Bergen en Henegouwen, P. M. P., Boonstra, J., Rijksen, G. (1997) Epidermal growth factor-induced activation and translocation of c-Src to the cytoskeleton depends on the actin binding domain of the EGF receptor. Biochim. Biophys. Acta 1359,211-221[Medline]
-
Diakonova, M., Payrastre, B., van Velzen, A. G., Hage, W. J., van Bergen en Henegouwen, P. M. P., Boonstra, J., Cremers, F. F. M., Humbel, B. M. (1995) Epidermal growth factor induces rapid and transient association of phospholipase C-1 with EGF receptor and filamentous actin at membrane ruffles of A431 cells. J. Cell Sci. 108,2499-2509[Abstract]
-
Zigmond, S. H. (1996) Signal transduction and actin filament organization. Curr. Opin. Cell Biol. 8,66-73[Medline]
-
Rebillard, M., Leibovitch, S., Julien, M., Talha, S., Harel, L. (1987) Early stimulation by EGF plus insulin of rRNA, c-fos, and actin mRNA expression, inhibition by cytochalasin D. Exp. Cell Res. 172,432-438[Medline]
-
Zambetti, G., Ramsey-Ewing, A., Bortell, R., Stein, G. (1991) Disruption of the cytoskeleton with cytochalasin D induces c-fos gene expression. Exp. Cell Res. 192,93-101[Medline]
-
Pavlath, G. K., Shimizu, Y., Shimizu, N. (1993) Cytoskeletal active drugs modulate signal transduction in the protein kinase C pathway. Cell. Struct. Funct. 18,151-160[Medline]
-
MacLean-Fletcher, S., Pollard, T. D. (1980) Mechanism of action of cytochalasin B on actin. Cell 20,329-341[Medline]
-
Rijken, P. J., van Hal, G. J., van der Heyden, M. A. G., Verkleij, A. J., Boonstra, J. (1998) Actin polymerization is required for negative feedback regulation of epidermal growth factor-induced signal transduction. Exp. Cell Res. 234,254-262
-
Davis, R. J. (1988) Independent mechanisms account for the regulation by protein kinase C of the epidermal growth factor receptor affinity and tyrosine protein kinase activity. J. Biol. Chem. 263,9462-9469[Abstract/Free Full Text]
-
Ito, M., Tanabe, F., Sato, A., Ishida, E., Takami, Y., Shigita, S. (1989) Possible involvement of microfilaments in protein kinase C translocation. Biochem. Biophys. Res. Commun. 160,1344-1349[Medline]
-
Sakisaka, T., Itoh, T., Miura, K., Takenawa, T. (1997) Phosphatidylinositol 4,5-bisphosphate phosphatase regulates the rearrangement of actin filaments. Mol. Cell. Biol. 17,3841-3849[Abstract]
-
Shariff, A., Luna, E. J. (1992) Diacylglycerol-stimulated formation of actin nucleation sites at plasma membrane. Science 256,245-247[Abstract/Free Full Text]
-
Lassing, I., Lindberg, U. (1988) Specificity of the interaction between phosphatidylinositol 4,5-bisphosphate and the profilin:actin complex. J. Cell Biol. 37,255-267
-
Janmey, P., Hvidt, S., Käas, J., Lerche, D., Maggs, A., Sackmann, E., Schliwa, M., Stossel, Y. P. (1994) The mechanical properties of actin gels. J. Biol. Chem. 269,32503-32513[Abstract/Free Full Text]
-
Wachsstock, D. H., Schwartz, W. H., Pollard, T. D. (1994) Crosslinker dynamics determine the mechanical properties of actin gels. Biophys. J. 66,801-807[Medline]
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ABSTRACT
INTRODUCTION
