Chapter B-III

Microgravity decreases c-fos induction and SRE activity

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

SUMMARY

Several studies have shown that altered gravity conditions influence mammalian cell growth and differentiation. The molecular mechanisms underlying these effects however remain relatively obscure. In this paper we show that microgravity reached in a sounding rocket strongly decreases epidermal growth factor (EGF) induced expression of the proto-oncogenes c-fos and c-jun, that are both implicated in the regulation of proliferation and differentiation. Decreased activity of the serum response element (SRE), present in the c-fos promoter - enhancer region, is probably responsible for the decrease in EGF-induced c-fos expression. In addition, we show that gravity alterations differentially modulate distinctive signal transduction pathways, indicating that gravity dependent modulations of mammalian cell proliferation are unlikely to be caused by a-specific stress responses of the cell.

INTRODUCTION

During the last decade life science research under altered gravity conditions has become a field of increasing interest. Although the mechanism of gravity sensation in plant cells is relatively well understood (Halstead and Dutcher, 1987), the knowledge of effects of altered gravity on animal cells is still rather limited. Extensive studies of gravity effects on mammalian cells were performed by Cogoli and coworkers, who showed that mitogenic stimulation of human lymphocyte cultures by the plant lectin concanavalin A (con A) is almost completely depressed under microgravity (Cogoli et al., 1984, Bechler et al., 1986), and that hypergravity enhances the stimulation of lymphocytes by con A (Lorenzi et al., 1986) as well as the proliferation of other mammalian cells (Tschopp and Cogoli, 1983).

How gravity alterations affect cell proliferation remains to be established. Possible clues for the underlying molecular mechanisms have been obtained from recent studies indicating that gravity may exert its effect by modulating the expression of growth regulatory genes, such as proto-oncogenes. Hypergravity appears to stimulate the proliferation of human HeLa cells through a reduction of the G1 phase duration, and enhances the expression of the c-myc proto-oncogene in these cells (Kumei et al., 1989), a gene whose product is known to play an important role in cellular proliferation (Kelly and Siebenlist, 1986). We have demonstrated that in human A431 cells EGF induced expression of the c-fos proto-oncogene is significantly decreased under simulated weightlessness conditions, whereas it is enhanced by hypergravity (de Groot et al., 1990a). In addition we found that EGF induced cell rounding is modulated by gravity changes (Rijken et al., 1990). Taken together these observations suggest that gravity may affect the intracellular signalling pathways activated by mitogenic stimuli such as growth factors, ultimately resulting in the modulation of proto-oncogene expression.

The gene products of the c-fos and c-jun proto-oncogene family are known for their prominent role in cell proliferation (reviewed in Imler and Wasylyk, 1989) and differentiation (Müller and Wagner, 1984; de Groot et al., 1990b). Their expression is usually rapidly induced by growth factors and can be induced also by a variety of agents that bypass the receptor and mimmick the partial activation of signal transduction pathways (for a review, see Verma and Sassone-Corsi, 1987). Examples of these latter are phorbol esters (e.g. TPA), Ca²⁺ (e.g. A23187) and agents that raise the intracellular concentration of cAMP (e.g. forskolin).

In this study we demonstrate that in A431 cells EGF induced c-fos and c-jun expression is strongly repressed in real microgravity, probably as a result of changes in promoter-enhancer activity. In addition we show that simulated microgravity differentially modulates distinctive signal transduction pathways, since under these conditions EGF and TPA induced c-fos expression is decreased whereas A23187 and forskolin induced c-fos expression is not.

MATERIALS AND METHODS

Cells and plasmids

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. As probe for Northern hybridization studies a 0.8 kb Pst1 fragment of v-fos (Curran et al., 1982) was used. Chloroamphenicol acetyltransferase (CAT) reporter plasmids including pSV2CAT have already been described (Gorman et al., 1982). The thymidine kinase promoter from herpes virus (positions -109/+57) was fused to the CAT structural gene and used as a background for the analysis of SRE sequences in the transfection experiments. SRE/DSE₃tkCAT is described elsewhere (de Groot et al., 1990c).

RNA isolation and Northern blotting

Total cellular RNA was isolated by the guanidine isothiocyanate-caesium chloride method (Chirgwin et al., 1979). 15 µg of total RNA was denatured for 10 min at 68⁰C in 50% (v/v) formamide, 2.2 M formaldehyde, 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS) pH 7.0, 5 mM sodium acetate, 1 mM ethylenediaminetetraacetic acid (EDTA), separated through 0.8 % agarose /2.2 M formaldehyde gels, and subsequently transferred to nitrocellulose filters (BA 85, Schleicher & Schuell) in 20X standard sodium citrate (SSC). RNA was immobilized by baking at 80⁰C for 2 hr under vacuum. Hybridization was performed in 50% formamide, 5X SSC, 50 mM sodium phosphate pH 6.8, 10 mM EDTA, 0.1% NaDodSO4, 0.1 mg of sonicated salmon sperm DNA per ml, 2X Denhardt solution (1X Denhardt solution contains 0.02% bovine serum albumin, 0.02% ficoll, 0.02% polyvinylpyrrolidone) at 42^oC overnight. ³²P-labeled probes were generated using a multiprime DNA labeling kit (Amersham). After hybridization and washing, filters were exposed to Kodak XAR-5 film at -70^oC using intensifying screens.

RNase Protection Analysis

RNase protection analysis was performed according to Melton et al. (1984). 1-2 µg of total cellular RNA was hybridized to ³²P-labeled complementary RNA probes derived from the human c-fos gene (Van Straaten et al., 1983), the human c-jun gene (Angel et al., 1988) and the human .²-2-microglobulin gene (Suggs et al., 1981). 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.

DNA transfection and transient expression assays

A431 cells were plated in DMEM-Bic/7.5% FCS at 4.10⁶ cells per 75 cm² tissue culture flask 24 hrs prior to transfection. Two hours before transfection, the flasks recieved fresh medium. Cells were incubated for 20-24 hrs with calcium phosphate precipitated DNA's (10-20 µg plasmid per flask), followed by trypsinization of the cells and replating on clinostat coverslips. 24 hours later, the medium was replaced for DMEM-Bic without FCS. After incubation for 2 hours, the cells were mounted in clinostat tubes, rotated and stimulated with or without EGF. After harvesting the cells and measuring protein concentrations, CAT activity was determined. 20 µg of cell extract was used for CAT assays (2 µg for pSV2CAT transfected cells). 1 µg of pSV2Apap DNA was always included to serve as internal control to correct for possible variations in the transfection efficiency. PAP assays were performed as described by (Henthorn et al., 1988). CAT activity was determined as described (Gorman et al., 1982) and was quantitated by liquid scintillation counting of TLC plate ¹⁴C spots.

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 37^oC.

MASER-3 sounding rocket experiment

A431 cells were cultured on coverslips and mounted into the CIS-1 plunger box experiment units (CCM, Nuenen, see Fig. 1). 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 37^oC 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 plunger A (see fig. 1). After 6 minutes the cells were washed with PBS (plungers B and D) and lyzed in guanidine isothiocyanate (plunger E). After recovery of the payload, RNA was isolated as described above.

RESULTS

Previously we have shown that EGF-induced expression of the c-fos proto-oncogene is significantly decreased under simulated hypogravity conditions (de Groot et al., 1990a). To investigate whether this effect also occurs in real microgravity, we performed an experiment in the CIS-1 module flown on the MASER-3 sounding rocket (Swedish Space Cooperation, April 1989). A431 cells were cultured in CIS-1 plunger box experiment units (Fig. 1) and stimulated with EGF (100 ng/ml) or medium alone (-EGF) for 6 min. after microgravity was reached in the rocket. Following recovery of the experiment units, RNA was isolated from the lyzed cells and analyzed for c-fos transcripts by RNase protection. As shown in figure 2, no c-fos transcripts were detected in unstimulated (-EGF) cells neither in the 1-G control experiment (ground), that was performed simultaneously with the flight experiment, nor in the microgravity samples (flight). By contrast, the 1-g samples that were stimulated with EGF for 6 minutes showed strong expression of the c-fos gene, which was decreased by about 50% in the cells that were treated with EGF in microgravity.

Since the product of the c-fos gene is known to form a functional heterodimeric complex with the product of the c-jun proto-oncogene (reviewed in Busch and Sassone-Corsi, 1990), the expression of the c-jun gene was studied. As shown in figure 2, c-jun expression was not detectable in unstimulated cells, whereas it was decreased by about 50% in the EGF treated flight sample as compared to EGF treated 1-g control cells. Expression of the .²-2-Microglobulin gene, a gene that is not modulated by EGF treatment, was constant in all flight and ground samples, indicating that the observed decrease in expression of c-fos and c-jun is specific for EGF induced signal transduction in these cells.

The induction of c-fos by EGF is mediated by the serum response element (SRE) present in the 5' regulatory region of the c-fos gene (Gilman et al., 1986; Treisman, 1986; Greenberg et al., 1987). This sequence binds at least three different regulatory proteins, of which p67-SRF (serum response factor) is probably mediating the effects of serum and EGF on c-fos expression (Treisman, 1987). To assess whether the observed effects of microgravity on EGF induced c-fos expression are caused by a modulation of SRE activity, we coupled three copies of the c-fos SRE to a heterologous promoter (HSV thymidine kinase, TK) and the bacterial chloroamphenicol acetyl transferase (CAT) gene. A431 cells were transfected with this construct (named FOS in figure 3) and replated on clinostat slides after 24 hours. The slides were mounted on a fast rotating clinostat after attachment of the cells for 24 hours and rotated at 60 rpm for another 2 hours. Hereafter, the cells were either treated with EGF (100 ng/ml) or medium alone for 8 hours. Collection of the cells and assaying for CAT activity was performed as described in materials and methods. As shown in figure 3A, no CAT activity was detected in cells treated with medium alone, while treatment with EGF leads to a strong enhancement of CAT activity. Comparison of EGF treated cells that were rotated on the clinostat (OG) and cells cultured in fixed clinostat tubes (1G) shows that simulated hypogravity decreased EGF-induced SRE activity. The activity of the SV40 promoter, that is transcriptionally active in these cells, was not modulated by simulated hypogravity, indicating that the decrease is specific for EGF induced SRE activity. Quantification of CAT activity of 4 independent experiments shows that EGF induced SRE activity (FOS in figure 3B), but not the activity of the SV40 promoter, is decreased by about 30% under simulated hypogravity conditions (Fig. 3B). These results show that the observed decrease in EGF induced c-fos expression is most likely caused by a decrease in SRE activity.

A variety of different signal transduction pathways lead to rapid increase of c-fos expression (reviewed in Verma and Sassone-Corsi, 1987). To examine if the observed modulations of c-fos expression by gravity changes are specific for EGF induced signal transduction, we studied the effects of simulated hypogravity on c-fos expression induced by the phorbol ester TPA, the Ca2+ ionophore A23187 and the activator of protein kinase A, forskolin. Cells were cultured for 40 hours on clinostat slides before mounting on a fast rotating clinostat. After rotation at 60 rpm for 2 hours, the cells were treated with EGF (100 ng/ml), TPA (100 ng/ml), forskolin (10 µM), A23187 (2.5 µM) or medium alone (CON) for 15 minutes. RNA was isolated and analyzed for c-fos expression by Northern blotting. As shown in figure 4A, both EGF- and TPA-induced c-fos expression was decreased under hypogravity conditions (OG in the figure), whereas forskolin- and A23187-induced c-fos expression remained constant. Scanning of the autoradiographs of 4 independent experiments shows that both EGF and TPA induced c-fos expression are significantly decreased in hypogravity (25% and 30% resp.), while no significant differences were found with A23187 or forskolin. These results clearly demonstrate that only a subset of signal transduction pathways that lead to c-fos induction is sensitive to gravity alterations, and are encouraging for further exploration of the effects of altered gravity conditions on mammalian cell proliferation and intracellular signal transduction.

DISCUSSION

Life science research under altered gravity conditions is important in determining the capacity of cells and organisms to cope with the problems of gravitational stress. Moreover it might lead to clues on the functioning of cells and organisms under normal gravity conditions. In this report we show that EGF induced expression of the c-fos and c-jun proto-oncogenes is severely decreased in real microgravity, extending our results from previous studies in simulated hypogravity (de Groot et al., 1990a). At least for the c-fos gene this effect is probably caused by decreased activity of the SRE located in its 5' regulatory sequences. Studying other agents that induce c-fos expression clearly shows that only a subset of signal transduction pathways is sensitive to gravity changes.

A recent study has shown that hypergravity caused by centrifugation enhances the expression of the c-myc proto-oncogene in proliferating HeLa cells in the abscence of EGF (Kumei et al., 1989). Although no changes in c-fos expression were found in these cells, our results are probably in agreement with this study since we only detect changes in c-fos expression in cells stimulated with growth factors or phorbol esters and not in unstimulated resting (no FCS, see fig. 2 and 4A) or proliferating cells (7.5% FCS, unpublished data). In addition we previously showed an enhancement of c-fos expression in hypergravity in EGF treated, but not in untreated A431 cells (de Groot et al., 1990a). The molecular basis of the differences between c-fos and c-myc sensitivity to gravity changes remains to be determined, but may involve the high constitutive expression of c-myc as opposed to the very low basal level expression of c-fos in unstimulated cells.

EGF induced c-fos expression is mediated by the SRE present in the c-fos promoter-enhancer region (see fig. 5 and Gilman et al., 1986; Treisman et al., 1986; Greenberg et al., 1987). This sequence binds at least three different proteins, of which p67-SRF is thought to mediate the effects of serum and EGF on c-fos expression (Treisman et al., 1987). Treatment of cells with EGF or serum leads to rapid phosphorylation of SRF, a process likely to be important in the transcriptional activation of c-fos (Prywes et al., 1988). Our results demonstrate that activation of the SRE by EGF is decreased under simulated hypogravity conditions, and suggest that this decrease is the basis for the observed effects of microgravity of EGF induced c-fos expression. In this respect it is worthwile to mention that TPA induced c-fos expression, a process that we show to be decreased in hypogravity, is also mediated by the SRE (see fig. 5 and Gilman et al., 1988; Stumpo et al., 1988). By contrast, forskolin and A23187 effects on c-fos expression are not sensitive to gravity changes, while these agents exert their effects on c-fos expression through regulatory sequences distinct from the SRE (see fig. 5 and Fisch et al., 1987; Gilman et al., 1988). The mechanism by which SRE activity is decreased under hypogravity conditions remains to be determined, but one could speculate that EGF induced phosphorylation of p67-SRF might somehow be modulated by gravity changes.

A variety of different agents induce expression of the c-fos proto-oncogene. Our results show that EGF and TPA, but not forskolin and A23187 induced c-fos expression is decreased in hypogravity. EGF exerts its effects through binding to its plasma-membrane located receptor followed by the activation of an intracellular signal transduction cascade (reviewed in Carpenter, 1987). Interestingly, activation of protein kinase C (PKC), the receptor for the phorbol ester TPA, is a part of this signal transduction cascade (fig. 5). This suggests that PKC activity, or the activity of a downstream target of PKC might be sensitive to gravity alterations. By contrast, both EGF and A23187 treatment leads to a rise in intracellular Ca2+ (fig. 5), suggesting that this process or its downstream targets are probably not influenced by gravity changes. Forskolin raises the intracellular cAMP concentration via activation of protein kinase A (PKA). Until now, no second messengers are identified that are shared by both EGF and forskolin induced signal transduction (fig. 5), further implicating PKC or one of its targets in gravity dependent modulations of mammalian signal transduction.

The products of the c-fos and c-jun genes are known to form functional heterodimeric complexes (reviewed in Busch and Sassone-Corsi, 1990), and are implicated in the control of proliferation (reviewed in Imler and Wasylyk, 1989) and differentiation (Müller and Wagner, 1984; de Groot et al., 1990b). Our study shows that EGF induced expression of these genes is strongly reduced in microgravity. Since a number of studies have shown gravity dependent modulations of mammalian cell proliferation and differentiation (Cogoli et al., 1984; Bechler et al., 1986; Lorenzi et al., 1986; Duke, 1983), our findings suggest that gravity dependent effects on c-fos and c-jun expression are likely to be of major importance for the cells reaction to gravitational stress. Further study of the regulation of these and other regulatory genes will hopefully lead to a more complete understanding of the way gravity exerts its effects on mammalian cells.

ACKNOWLEDGEMENTS

The authors would like to thank all the people involved in the CIS-1 experiment on the MASER-3 sounding rocket. This work was supported by grants from the Space Research Organization Netherlands (SRON).

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FIGURE LEGENDS

Figure 1 - Schematic representation of the CIS-1 plunger box experiment unit.
The CIS-1 plungerbox experiment unit is a slight modification of the unit used in the FROGS experiment flown on spacelab D1 (Ubbels, 1987) and was designed and constructed by CCM (Nuenen, The Netherlands). A transverse section of the module is shown. A431 cells are cultured on thermanox coverslips (TC) and mounted in the central chamber (C). After activation of plunger (PL) A by melting of a heater wire (HW), the cells are treated with EGF or medium alone, which is present in the fluid compartment (FC). Washing of the cells is performed by activation of plungers B and D, while the cells are lyzed by activation of plunger E. Plunger E is shown in the activated position. The route of the various solutions is indicated by arrows. After recovery, the lyzed cells are collected from chamber C and RNA is isolated.

Figure 2 - Microgravity decreases EGF-induced c-fos and c-jun expression.
A431 cells were cultured on thermanox coverslips, mounted in the CIS-1 plunger box experiment units and assembled in the CIS-1 module on the MASER-3 sounding rocket. As soon as microgravity was reached, the cells were treated with EGF (+EGF, 6 min) or with medium alone (-EGF, 6 min). As a control one sample was lyzed immediately after launch (-EGF, 0 min) to study the effects of the high g-values reached during the launch of the rocket. Simoultaneously a 1-g reference experiment (ground) was performed identically to the microgravity experiment (flight). After all samples were washed and lyzed, RNA was isolated and analyzed for c-fos, c-jun and .²-2-microglobulin transcripts by RNase protection. Autoradiography was for 16 hours.

Figure 3 - Hypogravity decreases SRE activity.
A - A431 cells cultured in 75 cm² flasks were transfected with 10 µg of a construct containing three copies of the c-fos serum response element (SRE, Treisman, 1986) coupled to the HSV-thymidine kinase promoter and the bacterial CAT gene (FOS in the figure) by Ca₃(PO₄)₂ precipitation. Control cells were transfected with an SV40 driven CAT gene (pSV2CAT, Gorman et al., 1982). After 24 hours the cells were trypsinyzed and replated on thermanox coverslips for 24 hours. The cells were mounted on a fast rotating clinostat and rotated for 2 hours at 60 rpm at 37^oC. EGF (+EGF) or medium alone (-EGF) was added and after rotation in the clinostat for 8 hours the cells were collected and CAT activity was determined on TLC plates. 20 µg of cell extract was routinely used for CAT assays (2µg for pSV2CAT transfected cells). Control cells were incubated identically in non-rotating fixed clinostat tubes (1G in figure).
B - The CAT activity of 4 independent experiments as in A was quantified by liquid scintillation counting of ¹⁴C TLC spots. Error bars indicate standard deviation.

Figure 4 - Differential sensitivity of discrete signal transduction pathways for hypogravity.
A - A431 cells were cultured on thermanox coverslips for 48 hours before mounting on a fast rotating clinostat. Cells were rotated at 60 rpm (0G in figure) or cultured in fixed tubes for 2 hours before addition of EGF (100 ng/ml), TPA (100 ng/ml), forskolin (Fors, 10 µM), A23187 (A23, 2.5 µM) or medium alone (CON). After 15 minutes the cells were lyzed and RNA was isolated. c-fos expression was determined by Northern blotting as described in materials and methods. Autoradiography was for 24 hours. B - The results of 3 independent experiments as in A were quantified by scanning of the autoradiographs. Bars represent the ratio of c-fos expression in control cells (1G) and rotated cells (0G). Error bars represent the standard deviation.

Figure 5 - Schematic representation of different signal transduction pathways leading to enhanced c-fos expression.