Previously we found that low concentrations of ethylene present in seawater  significantly reduce the extent of apoptosis caused by starvation in primmorphs of the marine sponge S. domuncula . Primmorphs, which contain proliferating cells, are formed by aggregation of dissociated sponge cells . In invertebrate (sponge) cells, the mode of action of ethylene, a well-known growth hormone in plants (for a review, see ), is still uncertain but seems to be associated with calcium metabolism .
In plants, ethylene has important regulatory functions; its production can be elicited by various stress factors (for a review, see ), including stress by oxygen radicals [15, 16]. The molecular basis of the ethylene signal transducing system has been studied mainly in Arabidopsis (for reviews, see [17,18,19]). A specific receptor to which ethylene binds has been identified .
In view of our finding that ethylene responsive pathways exist in sponges, the phylogenetic oldest metazoan phylum, we examined if cells derived from higher Metazoa (human and animal cell lines) respond to ethylene too. This alkene was produced in the culture fluid by addition of ethephon , which hydrolyzes in aqueous solution at pH >3.5 under formation of ethylene .
Using the Fura-2 imaging method, we could demonstrate that all mammalian cell lines studied reacted to ethylene, generated from ethephon, by an increase in [Ca2+]i level. The rise of [Ca2+]i level occurred immediately after addition of ethephon to the medium, indicating that ethylene causes a fast effect on cell metabolism. However, the threshold value and the extent of the ethylene-induced effect on [Ca2+]i level strongly differed among the cells examined. NIH-3T3 fibroblasts and osteosarcoma SaOS-2 cells sensitively responded to lower concentrations of ethylene produced by 0.3 mM ethephon, while the effect on HeLa cells was rather weak but significant (at 1 and 2 mM ethephon). In addition the amount of cells responding to ethylene within the total cell population varied among the cell lines examined.
Exposure of mammalian cells kept under pressure (SaOS-2 cells) to ethylene gas, instead of ethylene generated by ethephon, caused also a strong but more protracted increase in [Ca2+]i level compared to ethephon addition, most likely due to the different kinetics of changes of concentration of dissolved ethylene. This result and the finding that H3PO4 generated during ethephon hydrolysis does not change [Ca2+]i level demonstrate that the effects observed are specifically induced by ethylene. In addition, ethephon displayed no effect on cell viability.
The increase in [Ca2+]i level induced by ethylene exposure may be associated with changes in cell proliferation. Therefore, as a measure of the proliferative activity of the cells, the Ki-67 labeling index was determined. The Ki-67 antigen, a dimeric, non-histone protein with a molecular weight of 345-395 kDa is specifically expressed by proliferating cells; it is absent in resting cells . Using the Ki-S5 antibody, we determined that the expression of Ki-67 was significanly increased following ethylene exposure, indicating that ethylene may activate cell proliferation. At this time point (10 h after treatment with ethylene) [Ca2+]i has recovered basal levels (not shown in Figure 2A,B,C).
The concentrations at which the ethylene-releasing agent ethephon induced a shift of [Ca2+]i level in mammalian cells are in the same range or close to that at which sponge cells reacted (1 mM ethephon). However, the increase in [Ca2+]i level induced by this compound in at least some mammalian cell lines was even stronger than in sponge cells. From these results we conclude that besides invertebrate (sponge) cells, mammalian cells are sensitive to ethylene. In addition, we could demonstrate that the response of sponge cells following ethylene exposure depends on the presence of Ca2+ in the surrounding medium; no effect was observed if this metal ion was absent in the external milieu.
At present it is unknown if ethylene binds to a membrane receptor in mammalian cells, and sponge cells too. Therefore, the mechanism of the effect of ethylene resulting in an increase in intracellular level of calcium, one important messenger in intracellular signal transduction pathways, is still unclear. In sponge cells a Ca2+/calmodulin-dependent protein kinase II is up-regulated after ethylene exposure . In mammalian cells, it is known that Ca2+ mediates the Ca2+/calmodulin-dependent protein kinase II cascade , resulting in prevention of apoptosis . The second gene which was found to be upregulated in sponges after ethylene exposure is the proposed ethylene-responsive gene, SDERR, which has been isolated from S. domuncula .
It should be noted that the minimal concentration of ethylene required to evoke the raise of [Ca2+]i level in mammalian cells (3.2 μM) is rather high compared to the threshold concentration in plants, which may be as low as 0.1 μl/liter; this corresponds to an aqueous solution of 0.65 nM ethylene at 25°C . Therefore it cannot be excluded that the ethylene-induced effects in mammalian cells are non-specific due to the anesthetic effect of the gas which is not mediated by a receptor. Future studies have to show whether animal cells possess a specific receptor for ethylene gas as found in plant cells . These studies are necessary to demonstrate the physiological significance of the observed effects.
In summary, our results show that mammalian cells respond to ethylene with an increase in [Ca2+]i and cell proliferation (increased expression of cell-cycle-associated Ki-67 protein). In sponges the effects of ethylene on cell physiology are associated with an upregulation of an ethylene-responsive gene, SDERR . At present, it is unknown if similar proteins exist also in cells from higher animals and humans.