Epidermal growth factor (EGF) activates a well-
characterised signal transduction cascade in human A431
epidermoid carcinoma cells. This activation leads to increased
cell proliferation in most cell types. Among the early responses
evoked by EGF are receptor clustering, cell rounding and early
gene expression. The influence of gravity on EGF-induced EGF
receptor clustering and early gene expression, as well as actin
polymerisation, actin organisation and cell rounding have been
investigated.
EGF-induced c-fos and c-jun expression
decreased in microgravity. This was caused by the EGF receptor
and protein kinase C mediated signal transduction pathways, but
not by the Ca²+ or protein kinase A pathways. In
contrast, neither the binding of EGF to the receptor nor receptor
clustering was modulated under µg conditions. The relative
filamentous actin content was enhanced under µg and the
actin filament organisation was altered. Interestingly, the
microtubule and keratin organisation showed no difference under
µg.
A number of studies have indicated that gravity affects mammalian cell growth and differentiation.¹,² To study the effect of variations in gravity on human cells at the molecular level, the well-characterised epidermal growth factor (EGF)-induced signal transduction in A431 epidermoid carcinoma cells was used as a model system. EGF is a member of a family of polypeptide growth factors, involved in the control of mammalian cell growth and differentiation. It is a low molecular weight polypeptide which enhances the proliferation of various cell types both in vivo and in vitro. EGF acts by binding to a specific transmembrane receptor. This receptor is a glycoprotein with an extracellular EGF-binding domain and an intracellular protein tyrosine kinase domain. EGF binding to the receptor induces a dimerisation or clustering of part of the EGF receptor population. Several studies have indicated that receptor dimerisation leads to kinase activation. The subsequent signal transduction pathways activated by EGF include the phosphatidylinositol pathway and the ras pathway. These pathways lead to the induction of early gene expression such as the proto- oncogenesc-fos and c-jun. In addition, the EGF receptor activation leads to an increase in actin polymerisation and a drastic reorganisation of the actin microfilament system, leading to a rounding-up of the cells.³
To determine the influence of µg on EGF-induced signal transduction, experiments were performed during the MASER 3-6 flights, during which the effect of µg was studied on EGF- binding, EGF receptor clustering, actin polymerisation, cell morphology and early gene expression.
Cell culture
A431 human epidermoid
carcinoma cells were grown in Dulbecco modified essential medium
(DMEM) supplemented with 7.5% foetal calf serum (FCS) in a 7%
humidified atmosphere. For the experiments performed in the MASER
sounding rockets, the cells were cultured on coverslips and
mounted in Cells in Space (CIS) plunger box units as described
in ref. 10.
EGF binding studies
EGF binding was
determined by using 125 I-EGF (2 ng/ml) as described
in detail in ref. 4.
EGF receptor distribution
The EGF
receptor distribution was determined by the label fracture
method,5 using immunogold labelling as described in
detail in ref. 6.
Actin polymerisation and organisation The actin polymerisation and organisation was studied using fluorescent-labelled phalloidin with conventional and confocal laser scanning microscopy, as described in detail in refs. 6 & 7.
Early gene expression
The expression of
c-fos and c-jun was determined by the RNAse
protection assay as described in ref. 9.
Early gene expression
An excellent end-
point to monitor possible gravity effects on EGF-induced signal
transduction is provided by the EGF-induced expression of so-
called immediate early genes. These genes are the first to be
transcribed after stimulation of cells with growth factors such
as EGF. Examples are the c-fos, c-jun and c-myc
genes.
Experiments performed during the MASER 3-4 flights demonstrated that µg suppressed EGF-induced c-fos and c-jun expression (Fig. 1). On the other hand, the expression of the constitutively expressed ß-2 microglobulin gene, which is not modulated by EGF, remains unaffected.9 Quantitative analysis shows that c-fos and c-jun expression in µg is reduced by approximately 50% compared to the normal gravity control samples. These data demonstrate that µg inhibits expression that is specific for EGF-induced signal transduction.
Fig. 1. Microgravity decreases EGF-induced c-fos and c-jun
expression. As soon as µg was attained, A431 cells were
treated with EGF (+EGF) for 6 min or with buffer alone (-EGF) for
0-6 min. To test for possible launch effects, gene expression was
determined in samples lysed at the beginning of the µg phase
(Flight, -EGF, 0 min). RNA was isolated and analysed for c-fos,
c-jun and ß-2 microglobulin expression.
EGF binding
One likely explanation for
the observed inhibition of gene expression may well be that
µg leads to decreased binding of EGF to the cell surface-
located EGF receptor. EGF binding was therefore studied during
the MASER 5-6 flights, using125 I-EGF. These
experiments clearly showed that µg did not influence the
binding properties of the receptors (Fig. 2). Therefore, we
conclude that µg inhibits EGF-induced signal transduction
downstream of the initial activation, i.e. the binding of EGF to
its receptor.
Fig. 2. 125 I-radiolabelled EGF was allowed to bind
to the cell surface EGF receptor population for 6 min (A). Cells
were subsequently fixed, lysed and bound EGF was determined in
cpm/g of cellular protein. A similar experiment was performed
under µg during MASER 5 (B).
EGF receptor distribution
One of the
first cellular responses following binding of EGF to the receptor
is a redistribution of the cell surface EGF receptor population,
leading to receptor clustering.5 Electron microscope
techniques allow direct viewing and quantitative analysis of the
receptor distribution at an ultrastructural level. It was
demonstrated during the MASER 3-4 flights that the receptor
distribution of cells in control and EGF-treated cells in µg
and under normal gravity were fully comparable (Fig. 3). Thus
µg influences EGF-induced signal transduction downstream of
EGF binding and EGF receptor redistribution, but upstream of
early gene expression.
Fig. 3. The effect of µg on EGF-induced EGF receptor
clustering: a quantitative analysis. A431 cells were brought into
µg and fixed to detect launch effects. Other samples were
treated for 5 min with EGF or buffer alone under µg
conditions. After flight the cells were immunogold-labelled,
frozen and freeze-fractured. The mean percentage monomers of
cells from two independent experiments of launch control (0-),
EGF-treated (5+) and control (5-) cells were normalised according
to the regressions of the percentage monomers on the particle
density as described in detail in ref. 6.
Microgravity-sensitive signal transduction
pathways
The observations described so far indicate
that specific signalling pathways are affected by µg. This
could mean that µg 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 it
can also be triggered by a variety of agents that mimic the
partial activation of signal transduction pathways but bypass the
EGF receptor. Examples of such agents are the phorbol ester PMA
(activates protein kinase C), the calcium ionophore A23187
(mimics the EGF-induced increase in the intracellular free
calcium concentration) and forskolin (raises the intracellular
cyclic AMP concentration).
During the MASER 4-5 flights, A431 cells were activated respectively with EGF, PMA, A23187 and forskolin. Quantitative analysis of the c-fos and c-jun expression demonstrated that both EGF- and PMA-induced expressions were strongly inhibited under µg, while the forskolin- and A23187-induced gene expressions were not affected (Table 1). As EGF and PMA are known to be activaters of PKC, these data indicated that the cellular response to PKC-mediated signal transduction is a molecular target for µg.10
Table 1. EGF-induced c-fos and c-jun expression in µg. Early gene expression was determined by the RNAse protection assay as described in ref. 10. The ratios of gene expression under normal and µg conditions were determined. NC: not calculated; ND: not done. ß-2 microglobulin expression was determined as an internal reference.
Stimulus c-fos c-jun ß-2 microglobulin ----------------------------------------------------- control 0 NC NC 1.05 control 6 NC NC 1.15 EGF 1.90 2.25 0.95 PMA 1.35 2.05 0.95 A23187 1.05 0.95 1.05 forskolin 0.95 ND ND
The actin microfilament system
The former
observations indicate that specific signalling pathways are
affected by µg. This could mean that µg affects a
limited number of gravity-sensitive cellular targets. The actin
microfilament system is a possible candidate for mediating
gravity effects in A431 cells. This assumption is based on EGF-
induced cell rounding in A431 cells, a process dependent on actin
polymerisation, being enhanced in simulated µg.8
Preliminary data obtained during the MASER 5-6 flights indicate
that µg causes increased actin polymerisation. The precise
role of actin as a gravity-sensitive cellular component has,
however, still to be established.
The authors would like to thank ESA for providing the CIS facility, everyone involved in the successful MASER 3-6 flights and SRON for financial support.
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