Chapter B-IV

Nuclear responses to protein kinase C signal transduction are Sensitive to gravity changes

Rolf P. de Groot, Philip J. Rijken, Jeroen den Hertog, Johannes Boonstra, Arie J. Verkleij, Siegfried W. de Laat and Wiebe Kruijer

ABSTRACT

A number of studies have suggested that gravity changes may influence mammalian cell growth and differentiation. To obtain insight in the molecular mechanisms underlying these effects, we have studied immediate early gene expression in response to activation of cytoplasmic signal transduction under microgravity conditions. In this paper we show, that epidermal growth factor (EGF)- and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced expression of the c-fos and c-jun proto-oncogenes is decreased in microgravity, while no effect of gravity changes was observed on A23187-and forskolin-induced expression of these genes. The decrease in c-fos expression was not due to delayed kinetict under microgravity. These results demonstrate that gravity differentially modulates distinctive signal transduction pathways.

INTRODUCTION

Since the start of biological experiments in space in the early 70's, a number of studies have suggested that microgravity may have profound effects on cell growth and differentiation of both prokaryotic as well as eukaryotic cellular systems (for a review, see ref. 1). The most extensive study of gravity effects on mammalian cells was performed by Cogoli and coworkers, who have shown that mitogenic stimulation of human lymphocytes by the plant lectin concanavalin A (Con A) is almost completely abolished under microgravity conditions (2-3). Furthermore, they have shown that hypergravity enhances Con A-induced lymphocyte proliferation (4) as well as the proliferation of other mammalian cells (5).

Although the mechanism of gravity sensation in plant cells is relatively well understood (6), the molecular events underlying the effects of gravity alterations on mammalian cells are still largely unknown. However, recent studies have shown that expression of growth regulatory genes is modulated by gravity changes. In HeLa cells it was found that hypergravity enhances the expression of the c-myc proto-oncogene (7), a gene whose product is known to play an important role in cellular proliferation (8). Moreover, we have shown that EGF-induced expression of the c-fos and c-jun genes in human A431 epidermoid carcinoma cells is decreased under simulated (9) and real (10) microgravity conditions, while enhanced by hypergravity (9). These results show that gravity exerts its effect already at the early stages of the signal transduction cascade evoked by EGF.

The products of the c-fos and c-jun gene family are components of transcription factor AP-1, and play an important role in cell proliferation (for reviews see 11-12) and differentiation (13-14). The expression of these genes is rapidly induced by a wide variety of growth factors and other agents that selectively activates some components of signal transduction pathways (reviewed in 15-16). To further investigate the effects of microgravity on the expression of these genes, we performed an experiment in the CIS-2 module on the MASER-4 sounding rocket. Here we show that c-fos and c-jun induction by EGF and TPA was decreased under microgravity conditions, while no effect was found on c-fos and c-jun induction by forskolin and the Ca2+ ionophore A23817. Furthermore we show that the decrease in EGF-induced c-fos expression is not due to a delay in the kinetics of c-fos expression.

METHODS

Cells

Human A431 epidermoid carcinoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 7.5% foetal calf serum (FCS). One to four hours prior to stimulation, the medium was replaced for DMEM-Hepes without serum.

RNA isolation and RNase Protection Analysis

Total cellular RNA was isolated by the guanidine isothiocyanate-caesium chloride method (17). RNase protection analysis was performed according to Melton et al. (18). 1-2 ¼g of total cellular RNA was hybridized to 32P-labeled complementary RNA probes derived from the human c-fos gene (19), the human c-jun gene (20) and the human ²-2-microglobulin gene (21). After RNase digestion of the single strand transcripts, protected fragments of 110, 155 and 80 nucleotides are indicative for expression of c-fos, c-jun and ²-2-microglobulin respectively.

Clinostat experiments

For clinostat experiments, a portable fast rotating clinostat developed in cooperation with CCM (Centre for Construction and Mechanization, Nuenen, The Netherlands) was used. All experiments were performed at 60 rotations per minute at 37oC.

MASER-4 sounding rocket experiment

A431 cells were cultured on coverslips and mounted into the CIS-2 plunger box experiment units (CCM, Nuenen). The experiment units were assembled in boxes and loaded in the CIS-1 module (Dutch Space (former Fokker Space) and Systems, Amsterdam, The Netherlands) in the payload of the rocket. The temperature of the experiment units remained 37oC during the whole experiment due to active temperature control of the experiment boxes. After microgravity was reached in the rocket, the cells were stimulated with EGF (100 ng/ml) or medium alone (-EGF) by activation of a plunger. After 6 minutes the cells were lyzed in guanidine isothiocyanate by activation of another plunger. After recovery of the payload, RNA was isolated as described above.

RESULTS

We have previously shown that EGF-induced expression of the c-fos and c-jun proto-oncogenes was decreased under simulated and real microgravity conditions (9-10). To examine whether other signalling pathways leading to the expression of these genes were also sensitive to gravity changes, we performed an experiment on the MASER-4 sounding rocket. A431 cells were cultured on thermanox coverslips in "plunger box units" (10). After microgravity was reached in the rocket, cells were automatically stimulated with EGF, TPA, the Ca2+ ionophore A23187, forskolin or medium alone for 6 minutes, after which the cells were lyzed. RNA was isolated after recovery of the rocket and analyzed for the expression of c-fos and c-jun. A 1G-reference experiment was performed in parallel on the ground using identical hardware. Figure 1A shows that EGF- and TPA-induced c-fos expression was decreased under microgravity conditions (47 and 26%, respectively; Table I), while no effects of microgravity were observed on c-fos expression induced by A23187 or forskolin. No c-fos expression was detected in untreated cells (Con 0 and 6). The expression of the ²-2-microglobulin gene, that is not influenced by signal transduction, was not modulated by microgravity (Fig. 1A and table I), indicating that the observed effects of gravity changes on c-fos expression result from specific modulations of the signal transduction processes induced by EGF and TPA. Although these experiments were only performed in duplicate due to space limitations in the rocket, they are in agreement with previous results from experiments under simulated microgravity conditions reached in a fast rotating klinostat (9-10) as well as under real microgravity (10). When the expression of c-jun was studied, similar results were obtained. Figure 1B shows that EGF- and TPA-induced c-jun expression was decreased by microgravity (56 and 51%, respectively; Table II), while A23187-induced c-jun expression was not altered significantly (forskolin was not tested, since it does not induce c-jun expression in these cells). These results indicate that the effects of gravity on immediate-early gene expression do not result from a general cellular stress response, but are more likely caused by specific modulations of distinctive signal transduction pathways.

Since these experiments did not address the question whether under microgravity conditions the induction of c-fos and c-jun was decreased or was delayed compared to the 1G reference, we performed time-course experiments under simulated microgravity in the fast rotating klinostat. A431 cells cultured on thermanox coverslips were pre-rotated for 2 hours, after which EGF was added for 10 to 90 min. (maximum levels of c-fos expression are reached after 30 min., while after 90 min c-fos expression is decreased to about 20% of its maximal value) after which RNA was isolated and analyzed for c-fos expression. As shown in figure 2, the ratio of c-fos expression under 1G conditions and under simulated microgravity (0G) conditions was significantly larger than 1 for all the time points tested, indicating that simulated microgravity does not delay the kinetics of EGF-induced c-fos expression, but decreases the level of expression of this gene.

DISCUSSION

EGF exerts its effect through binding to its plasma-membrane located receptor, followed by a rapid activation of an intracellular signal transduction cascade (reviewed in 22). Since activation of protein kinase C (PKC), the natural receptor for TPA, is one of the key events in this signal transduction cascade, our results suggest that PKC or a PKC-modulated protein may be one of the cells targets for gravity alterations. By contrast, the raise in intracellular Ca2+ concentration induced by both EGF and A23187 is not likely to be modulated by gravity changes. Forskolin-induced changes in gene expression are mediated by protein kinase A (PKA), which does not share common second messengers with PKC in its signal transduction cascade. Interestingly, recent experiments by Limouse et al. show that the production of interleukins (IL-1 and IL-2) by T lymphocytes and monocytes in response to TPA is almost completely decreased under long-duration microgravity conditions (12 hrs), while IL production induced by cell-to-cell contacts was not gravity dependent (accompanying paper). Combined with our data, these results strongly suggest PKC or one of its down-stream targets in gravity-dependent modulations of mammalian signal transduction, although we did not observe a complete inhibition of TPA-induced gene expression, probably due to the short exposure to microgravity (6 min.).

At present, we can only speculate about the gravity-sensitive component in the PKC signalling pathway. However, we have previously shown that the activity of the c-fos serum response element (SRE) is decreased under simulated microgravity conditions (10). Interestingly, this element mediates c-fos induction by EGF as well as by TPA (23-26). By contrast, A23187 and forskolin induce c-fos expression through regulatory sequences distinct from the SRE (27-28). The SRE binds at least three different proteins, of which p67-SRF seems to be responsible for activating c-fos expression in response to EGF (29). Treatment of cells with EGF or serum leads to a rapid phosphorylation of SRF, which is suggested to be of major importance for transcriptional activation of c-fos (30). Since our results clearly demonstrate that SRE-dependent c-fos induction is sensitive to gravity alterations, it seems likely that PKC-activated events leading to phosphorylation of SRF might be one of the processes influenced by altered gravity conditions.

The products of the c-fos and c-jun genes are implicated in the regulation of mammalian cell proliferation and differentiation (11-14). A number of previous studies have demonstrated effects of gravity changes on cellular proliferation and differentiation (2-4, 31) It is therefore tempting to speculate that gravity-dependent modulations of c-fos and c-jun expression may play a role in these processes. In this respect it is noteworthy to mention that stimulation of lymphocyte proliferation by ConA, a process that is highly sensitive to gravity changes (2-4), is preceded by the induction of c-fos and c-myc (R.P. de Groot, unpublished results). More experiments are however needed to further elucidate the mechanism by which gravity influences early gene expression responses to signal transduction, and to determine whether these changes are critical for the cellular response to gravitational stress.

Acknowledgements

The authors would like to thank all persons involved in the CIS-2 experiment on the MASER-4 sounding rocket. This work was supported by grants from the Space Research Organization Netherlands (SRON).

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Figure 1 - Gravity alterations differentially modulate distinctive signal transduction pathways.
A - A431 cells cultured in the sounding rocket (0G) or in the 1G reference setup (1G) were treated for 6 minutes with EGF (100 ng/ml), A23187 (2.5 ¼M), TPA (100 ng/ml), forskolin (10 ¼M) or medium alone (CON 6), after which the cells were lyzed. As control to determine the effect of high G-levels reached during the launch of the rocket, cells were lyzed directly after reaching microgravity in the rocket (CON 0). All experiments were performed in duplicate. RNA was isolated and analyzed for c-fos and ²-2-microglobulin (²2M) expression by RNase protection. B - A subset of RNA samples from A were analyzed for c-jun expression.


Figure 2 - Simulated microgravity does not influence the kinetics of c-fos expression.
A431 cells cultured in a fast rotating klinostat (0G) or in the 1G reference setup were treated for the indicated times with EGF. RNA was isolated and analyzed for c-fos expression. Protected fragments were cut out of the gel and counted in a liquid scintillation counter. Bars represent the 1G / 0G ratio of c-fos expression at the indicated time points after EGF addition, and are the mean of 6 independent experiments. Error bars represent the standard deviation.
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