Ethylene Modulates Gene Expression in Cells of the Marine SpongeSuberites domuncula and Reduces the Degree of Apoptosis*

Sponges (phylum Porifera) live in an aqueous milieu that contains dissolved organic carbon. This is degraded photochemically by ultraviolet radiation to alkenes, particularly to ethylene. This study demonstrates that sponge cells (here the demosponge Suberites domuncula has been used), which have assembled to primmorphs, react to 5 μm ethylene with a significant up-regulation of intracellular Ca2+concentration and with a reduction of starvation-induced apoptosis. In primmorphs from S. domuncula the expression of two genes is up-regulated after exposure to ethylene. The cDNA of the first gene (SDERR) isolated from S. domuncula encodes a potential ethylene-responsive protein, termed ERR_SUBDO; its putativeM r is 32,704. Data bank search revealed that the sponge polypeptide shares high similarity (82% on amino acid level) with the corresponding plant molecule, the ethylene-inducible protein from Hevea brasiliensis. Until now no other metazoan ethylene-responsive proteins have been identified. The second gene, whose expression is up-regulated in response to ethylene is a Ca2+/calmodulin-dependent protein kinase II. Its cDNA, SDCCdPK, encodes a M r54,863 putative kinase that shares 69% similarity with the corresponding enzyme from Drosophila melanogaster. The expression of both genes in primmorphs from S. domuncula is increased by ≈5-fold after a 3-day incubation period with ethylene. It is concluded that also metazoan cells, with sponge cells as a model, may react to ethylene with an activation of cell metabolism including gene induction.

Sponges (phylum Porifera) represent the phylogenetic oldest Metazoa that share a common ancestor with all multicellular animals; they lived already before the "Cambrian Explosion" at least 580 million years ago (for review, see Refs. [1][2][3][4]. Sponges possess most of the structural elements known from more complex Metazoa, e.g. adhesion molecules (galectin), adhesion receptors (receptor tyrosine kinase, integrin receptor, receptor(s) featuring scavenger receptor cysteine-rich domains) or elements involved in signal transduction pathways (G-proteins, Ser/Thr protein kinases) (for review, see Ref. 5). Additionally sponges display simple elements of an immune system related to that found in vertebrates, e.g. macrophage-derived cytokine-like molecules, the (2Ј-5Ј)oligoadenylate synthetase system, or a molecule very similar to the mammalian T-cell receptor (6) (for review, see Ref. 7).
Ethylene is one major alkene produced in seawater from oceanic dissolved organic carbon by photochemical reactions initiated especially by sunlight (ultraviolet radiation) (8 -10). The concentration of ethylene in filtered seawater was determined to be close to 100 pM (11) and remains almost constant during a period of 8 days in the dark (11). The concentration of dissolved organic carbon is similarly high (12) in the habitats of sponges. Because sponges are efficient benthic filter-feeders, some of which filter 24,000 liters kg Ϫ1 of sponge (13) every day, they are likely to take up large amounts of ethylene.
There have been only a few successful approaches to define the energy sources of sponges. It is generally assumed that sponges ingest particulate nutrients by phagocytosis (for review, see Ref. 14) or accumulate amino acids from the marine environment (15). However, until recently it was not possible to cultivate sponge cells by feeding them with particulate nutrients, e.g. bacteria or subcellular particles from them, and/or in media composed of amino acids or vitamins, only. Recently, a technique was established that allows single sponge cells to associate to "organ-like" aggregates, termed primmorphs; in this state the cells are able to divide (16). Evidence has been presented that some cells within the primmorphs undergo apoptotic death; subsequently the resulting cell fragments are very likely taken up via phagocytosis by those cells that express on their surface receptors composed of scavenger receptor cysteine-rich repeats (7,17). So far, the volatile products in the marine environment including ethylene have not been experimentally studied as a potential energy source for sponges.
The rationale of this study was to investigate the effect of ethylene on sponge metabolism. Ethylene is known to serve as an energy source for some bacteria, e.g. Paracoccus denitrificans (18), and to contribute to plant growth (for review, see Ref. 19). Recently we showed that starvation of Suberites domuncula as a whole and also of single cells results in the induction of apoptosis (20). To determine the effect of ethylene on sponge cells, primmorphs from S. domuncula, containing proliferating cells (16), were kept under pressure either in the absence or presence of ethylene to imitate natural conditions. It is striking that most sponges live in a species-specific depth in the marine environment. As an example, S. domuncula lives preferentially in a depth of 20 -25 m (21), suggesting that the pressure of the surrounding water is one important factor for its growth.
Here we show that ethylene reduces the extent of apoptosis caused by starvation. Two molecular markers for the effect of ethylene were chosen. As a first molecule, the sponge gene, related to the plant stress-induced gene HEVER, which was * This work was supported by grants from the Bundesministerium fü r Bildung und Forschung (Project Cell Culture of Sponges, project 03F0197A) and the International Human Frontier Science Program (RG-333/96-M). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) Y19159, Y19007.
‡ To whom correspondence should be addressed. found to be ethylene-responsive (22), was selected. This sponge gene was cloned; its expression is strongly induced after exposure of cells to ethylene. The second gene selected was the Ca 2ϩ /calmodulin-dependent protein kinase II (CaM kinase II), 1 which plays a central role in transduction of Ca 2ϩ signals in cells. In previous studies (23) it had been demonstrated that the intracellular concentration of Ca 2ϩ , [Ca 2ϩ ] i , changes rapidly in response to exposure of agonists for the metabotropic glutamate receptor. The CaM kinase II is activated by an increase of [Ca 2ϩ ] i (24) and also causes gene expression (25). Calmodulin has been shown to play a crucial role in integrinmediated signal transduction in sponges (26) via activation of CaM kinase II. 2 The expression of the CaM kinase II gene is up-regulated after exposure to ethylene.
The results presented in this study show for the first time that, among Metazoa, sponges are provided with a signaling cascade in which ethylene activates cell metabolism and gene expression.

EXPERIMENTAL PROCEDURES
Materials and Solutions-The sources of chemicals and enzymes used were given previously (26,27). The composition of Ca 2ϩ -and Mg 2ϩ -free artificial seawater was described earlier (28). Natural, Ca 2ϩ -, and Mg 2ϩ -containing seawater (SW) was obtained from Sigma (Deisenhofen, Germany).
Sponges-Specimens of the marine sponge S. domuncula (Porifera, Demospongiae, Hadromerida) were collected in the northern Adriatic near Rovinj (Croatia) and then kept in aquaria in Mainz (Germany) at a temperature of 17°C.
Dissociation of Cells and Formation of Primmorphs-The procedure for dissociation of sponge cells was described previously (16,29). Primmorphs, special aggregates, reassociated from single cells after transferring them into medium composed of SW (16,29), supplemented with 0.1% (v/v) of Marine broth 2216 (Difco). A suspension of 10 6 cells/ml is adjusted; after two days, primmorphs at least 1-mm in diameter (average, 2-3-mm) are formed. After 5 days, the primmorphs were used for the incubation experiments.
The primmorphs were kept under pressure of 1 physical atmosphere (atm) (Fig. 1). The pressure in the culture chamber was generated by air. In the studies using ethylene, reservoir I was filled with medium (SW containing Marine broth) and 5 M ethylene (adjusted from a stock solution of 1 mM ethylene). The solution was pumped at a rate of 1 ml/h through the culture chamber; the extruded medium was collected in reservoir II (Fig. 1A). The culture chamber (3.5 ϫ 1-cm) contained the primmorphs in 6 ml of medium (Fig. 1B).
Loading of S. domuncula Cells with Fura-2-AM and Measurement of Intracellular Calcium-Chambered coverglass incubation chambers (Lab-Tek, Nunc) were coated with poly-L-lysine (M r Ͼ 300,000; 0.1 mg/ml) as described (23,26). Loading was performed in the dark for 120 min in Ca 2ϩ -and Mg 2ϩ -free artificial seawater containing 10 -12 M Fura-2-AM and 1% bovine serum albumin. To determine the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ), cells on coverglass were transferred into the pressure chamber (Fig. 1C). After exposure for 3 h to 1 atm the cells were illuminated with 340-and 380-nm light from a mercury source (23,26). Ratios of sequential 340/380-nm excitation image pairs were compared with a standard curve for free Ca 2ϩ (30). Measurements were performed in a Ca 2ϩ -containing (10 mM) modified Locke's solution adjusted to seawater osmolarity (500 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO 3 , 5.6 mM glucose, and 10 mM HEPES, pH 7.4). Calibration was performed with the Fura-2 calcium imaging calibration kit according to the manufacturer's instructions (Molecular Probes, Leiden, The Netherlands). One ratio unit 340/380-nm corresponds to 143 nM [Ca 2ϩ ] i.
For the incubation experiments with ethylene, the culture medium in the chamber (diameter of the glass plates was 1 cm) was adjusted to 5 M ethylene.
Cell Death Assay-DNA fragmentation (TUNEL staining) was deter-mined using the in situ cell death detection kit (Roche Molecular Biochemicals). Dissociated cells were air-dried and fixed using 4% paraformaldehyde following the manufacturer's protocol. The TUNELstained cells were counterstained with propidium iodide (5 g/ml) and visualized by fluorescence microscopy. Polymerase Chain Reaction (PCR) Cloning of the Sponge Ethyleneresponsive Protein-The complete sponge cDNA, encoding the related ethylene-responsive protein, termed SDERR, was isolated from the S. domuncula cDNA library by PCR (27). The degenerate forward primer, directed against the conserved aa segments found in the sequences from 1 The abbreviations used are: CaM kinase II, Ca 2ϩ /calmodulin-dependent protein kinase II; DIG, digoxigenin; SW, Ca 2ϩ -and Mg 2ϩcontaining seawater; TUNEL, terminal dUTP nick-end labeling; aa, amino acid(s); PCR, polymerase chain reaction; bp, base pair(s); nt, nucleotide(s). 2  A, pressure was generated by air in the culture chamber. Medium was pumped into the culture chamber from reservoir I, filled with medium (and with ethylene if indicated) by a tube pump, and finally collected again in reservoir II. B, the culture chamber that contains the primmorphs has a diameter of 3.5 cm and is filled with 6 ml of medium; the tubes pumping the medium in (in) or releasing it out (out) are marked. C, pressure chamber for the determination of the changes of [Ca 2ϩ ] i in response to ethylene. The arrow marks the position where the coverglass was inserted into the pressure chamber. The tube and the pressure manometer used to adjust the pressure are shown. The objectives of the inverted microscope are focused on the cells from beneath the otherwise dark pressure chamber. The tubes through which the ethylene-enriched SW was injected into (in) and extruded from (out) the chamber are indicated. the ethylene-inducible protein from Hevea brasiliensis (aa 157 and aa 167 ; GenBank TM accession number Q39963) (22), the hypothetical 31.4-kDa protein C29B12.04 of Schizosaccharomyces pombe (aa 143 and aa 153 ; GenBank TM accession number O14027) and the hypothetical protein MTH666 from Methanobacterium thermoautotrophicum (aa 140 and aa 150 ; GenBank TM accession number O26762), 5Ј-GA(G/A)GGIGCIGCI-ATGATICGIACIAA(A/G)GGIGA-3Ј (where I ϭ inosine), in conjunction with the ZAPII 3Ј-end vector-specific primer T7 was used. The PCR reaction was carried out at an initial denaturation for 3 min at 95°C, then 32 amplification cycles at 95°C for 30 s, 67.5°C for 45 s, 74°C for 1.5 min, and a final extension step at 74°C for 10 min at 74°C. The reaction mixture was as described earlier (31). The fragment of 700-bp was used to isolate the cDNA from the library (32). The longest insert obtained was 1,106 nt (excluding the poly(A) tail). The clone was termed SDERR and was sequenced using an automatic DNA sequenator .
PCR Cloning of the Sponge Ca 2ϩ /Calmodulin-dependent Protein Kinase II-The cDNA, encoding the complete CaM kinase II, CCdPK-_SUBDO) SDCCdPK was isolated using the same strategy. Here, the forward primer 5Ј-GCIAA(A/G)GA(T/C)CTIATIAA(T/C)AAA/GATG-3Ј directed against one conserved region within the human CaM kinase II subunit ␦-2 (33) (aa 245 and aa 252 ) and an annealing temperature of 50°C were used. The 700-bp fragment was obtained and used for the isolation of the cDNA; the insert had a size of 1,714 nt.
Sequence Comparisons-The sequences were analyzed using computer programs BLAST (34) and FASTA (35). Multiple alignments were performed with CLUSTAL W version 1.6 (36). Phylogenetic trees were constructed on the basis of aa sequence alignments by neighbor-joining, as implemented in the "Neighbor" program from the PHYLIP package (37). The distance matrices were calculated using the Dayhoff peptidylglycine ␣-amidating monooxygenase matrix model as described (38). The degree of support for internal branches was further assessed by bootstrapping (37). The graphic presentations were prepared with GeneDoc (39).
Northern Blotting-RNA was extracted from liquid nitrogen-pulverized sponge tissue with TriZOL TM Reagent (Life Technologies, Inc.) as recommended by the manufacturer. 3 g of total RNA was electrophoresed through a formaldehyde/agarose gel and blotted onto a Hybond N ϩ membrane following the manufacturer's instructions (Amersham Pharmacia Biotech). Hybridization experiments were performed with the following probes: the Ϸ580-bp fragment of SDERR (nt 359 to nt 943 ) from S. domuncula, S. domuncula SDCCdPK (a segment of Ϸ530 bp was used; nt 294 to nt 821 ), and the S. domuncula ␤-tubulin 2 SDBTUB (Ϸ800 bp).
These probes were labeled with DIG-11-dUTP by the DIG DNA labeling kit. Hybridization was performed with the antisense DIGlabeled probes at 42°C overnight using 50% formamide, containing 5ϫ SSC, 2% blocking reagent, 7% (w/v) SDS, and 0.1% (w/v) N-lauroylsarcosine, following the instructions of the manufacturer (Roche Molecular Biochemicals). After washing DIG-labeled nucleic acid was detected with anti-DIG Fab fragments (conjugated to alkaline phosphatase) and visualized by the chemiluminescence technique using CDP-Star, the chemiluminescence substrate alkaline phosphatase, according to the instructions of the manufacturer.
To quantitate the signals of the Northern blots, the chemiluminescence procedure was applied (40). The screen was scanned with the GS-525 Molecular Imager (Bio-Rad). The relative values for the expressions of the ERR_SUBDO and CCdPK_SUBDO genes in S. domuncula cells were correlated with the intensities of the bands measured for the expression of the tubulin gene.

Induction of Apoptosis in Primmorphs after Starvation and the Effect of Ethylene-
The primmorphs generally contain a small fraction of apoptotic cells (approximately 8 Ϯ 3% of TUNEL-positive cells). If the primmorphs were cultured in SW lacking the supplement (Marine broth), the number of apoptotic cells increased and reached a value of 16 Ϯ 4% after 24 h and 29 Ϯ 5% after 48 h (Fig. 2). If primmorphs were kept under starvation (lack of Marine broth) but in the presence of 5 M ethylene, the number of apoptotic cells did not change significantly; values of Ϸ10 Ϯ 4% were measured.
Effect of Ethylene on the Intracellular Ca 2ϩ Concentrations in Cells from S. domuncula-Dissociated cells from S. domuncula were incubated with 5 M ethylene, and the [Ca 2ϩ ] i level was determined. The assays were performed with Ca 2ϩ -containing incubation solution. If the cells were incubated for 23 min in the absence of ethylene no significant change of the [Ca 2ϩ ] i was observed as checked by fluorescence (Fig. 3A, panels a-c). However, if the cells were treated with ethylene, an immediate shift of the fluorescence of most of the cells from blue to yellow/red is seen (Fig. 3B, panels a and b (5 min) and c (23 min)). The light microscopical aspects of the cells analyzed are given (Fig. 3, A, panel d and B, panel d).
A quantitative analysis of the changes of the [Ca 2ϩ ] i levels is summarized in Fig. 4. Cells not treated with ethylene did not show a change of the [Ca 2ϩ ] i level; a ratio value (340/380 nm) of Ϸ1.73 Ϯ 0.04 was measured. A significant shift of the ratio was measured if ethylene was added to the cells; it increased from 1.80 Ϯ 0.04 (time zero) to 1.90 Ϯ 0.02 (20 min after addition of ethylene) (Fig. 4).
Cloning of the Ethylene-responsive Protein from S. domuncula-The complete cDNA, encoding the ethylene-responsive protein from S. domuncula, termed SDERR, is 1,106-nt long and has a potential open reading frame (ORF) from the putative AUG initiation codon at nt 39 -41 to the stop codon at 957-959, that encodes a 306-aa long polypeptide (Fig. 5). The deduced aa sequence of the putative ethylene-responsive protein termed ERR_SUBDO has a putative size (M r ) of 32,704 and an isoelectric point (pI) of 5.87 (41). A bipartite nuclear targeting signature is present between aa 63 and aa 79 (Fig. 5). Northern blot analysis performed with the sponge SDERR clone as a probe yielded one prominent band of approximately 1.4 kilobases, confirming that a full-length cDNA was isolated (Fig. 6A).
A data bank search with the deduced aa sequence, ERR_SUBDO, revealed a high identity (similarity) to the plant sequence, the ethylene-inducible protein from H. brasiliensis (22)  2.0 kilobases (Fig. 6B). The deduced polypeptide comprises three autophosphorylation sites at Thr-283, Thr-302, and Ser-311, the autoinhibitory region (aa 269 and aa 299 ), which partially overlaps with the calmodulin-binding domain (aa 288 and aa 307 ) and the variable region, which spans in S. domuncula the region aa 311 to aa 370 (24). The Ϸ20 aa at the COOH terminus, which are present in the ␦-subunits of other CaM kinase II, are missing in the sponge sequence, whereas the Ser/Thr protein kinase active site (aa 129 and aa 141 ) and the ATP-binding signature (aa 17 and aa 25 ) are found as indicated in Fig. 7A.
Phylogenetic Analysis of Sponge CaM Kinase II-Until now no CaM kinase II enzymes have been described or cloned from evolutionary older phyla than from Porifera. The S. domuncula enzyme CaM kinase II with a M r of 54,863 belongs, based on its size, to the ␣-subunit of this kinase family (24). The closest similarity of the sponge protein was found to be the corresponding enzyme from Drosophila melanogaster, also an ␣-subunit CaM kinase II (42) with an identity (similarity) of Ϸ52% (Ϸ69%). An unrooted phylogenetic tree was constructed (Fig.  7B) with the distantly related peripheral plasma membrane protein CASK from Rattus norvegicus. The tree revealed that the sponge CaM kinase II forms the basis for the related enzymes from protostomians, D. melanogaster, Caenorhabditis elegans and deuterostomians, chicken, Xenopus laevis and human.
Levels of Expression of Sponge Ethylene-responsive Protein and CaM Kinase II in Dependence upon Ethylene-In the absence of ethylene, the expression of the gene encoding the ethylene-responsive protein is low (Fig. 6A). However, already after an incubation of 1 day in the presence of ethylene the expression of SDERR increased significantly (1.8-fold) and reached a maximum after 3 days (6.6-fold). A similar pattern is seen for the expression of CaM kinase II. The steady state level of this enzyme is low in the absence of ethylene and increased drastically after 3 (5.5-fold) to 5 days (6.1-fold) (Fig. 6B). In parallel blots, the level of expression of ␤-tubulin RNA from ethylene-treated primmorphs was determined; no significant differences were seen, confirming that the same amount of RNA was applied (Fig. 6C). DISCUSSION The Porifera lived before the Cambrian Explosion, a time at which presumably a lower oxygen content in the atmosphere existed than at present (43). Consequently, during that period a less dense ozone screen protected living organisms against the ultraviolet (UV) fluxes (44) and precluded the development of terrestrial life (45). Therefore, it can be assumed that the animals living in the marine environment were exposed to higher levels of alkenes, due to UV-mediated photochemical degradation of dissolved organic carbon than today. Based on this consideration it was not too surprising that the hypothesis that sponges react to alkenes with an increase of their metabolism could experimentally at least partially be supported.
In a first set of experiments it was shown, in accordance with earlier observations (20), that sponge cells react to starvation with an increased rate of apoptosis. Treatment of cells in seawater supplemented with low concentrations of ethylene significantly reduces the number of apoptotic cells in the primmorphs. This result hints at a beneficial anabolic effect is caused by this volatile product. Ethylene occurs in ripening plants in high concentrations that are not toxic for humans (46). However, in Metazoa no report has been presented that ethylene can be used as energy source or for any other physiological and metabolically favorable process. Therefore, the following experiments were performed to substantiate the assumption that ethylene stimulates cell metabolism in sponges.
Intracellular Ca 2ϩ is of pivotal importance for many biological processes, e.g. as stabilizer of intracellular structures or as second messenger in signal transduction pathways (47). Therefore, the determination of [Ca 2ϩ ] i in response to a given extracellular stimulus is a useful measure for cell response. Here we report that immediately after enrichment of the medium with ethylene the cells respond with a significant rise of [Ca 2ϩ ] i . This finding might indicate that this alkene causes an immediate effect on cell metabolism. Because no data are available in Metazoa about whether ethylene binds to a membrane receptor that might be coupled to a G-protein, it is too early to speculate about a possible effect of ethylene on a signal transduction pathway that releases Ca 2ϩ from intracellular stores.
In plants, ethylene very likely interacts with a putative ETR1 receptor, which displays similarity to both histidine kinase and response regulator domains of the bacterial twocomponent sensing system (48). Downstream within the signal transduction cascade, the CTR1 kinase has been identified (49). This enzyme belongs to the family of Ser/Thr protein kinases (50). CTR1 is differentially regulated by ethylene and air, a process which ultimately results in changed expression of various genes (51). Among those is the HEVER gene, which is induced in H. brasiliensis in response to ethylene (22). The deduced aa sequence of the sponge-related gene, SDERR, shares high similarity to the plant molecule (Ͼ80% similar aa with the same physico-chemical properties). The expression of the sponge ethylene-responsive gene is strongly up-regulated 1 day after ethylene exposure. It is the first ethylene-responsive gene that has been described in Metazoa, which may be due to the fact that either this gene is missing in higher metazoans or it has not yet been searched for.
The modulatory effect of ethylene on gene expression in sponge cells is not restricted to SDERR alone. It is shown that ethylene also causes an increased expression of CaM kinase II in S. domuncula cells. The deduced aa sequence of the enzyme shares the characteristic signatures for this family of kinases (24). The sponge CaM kinase II may be preliminarily classified to the ␣-subunits because of its size; it comprises 483 aa residues in comparison with the 478-aa-long ␣-subunits of higher metazoans, whereas the other subunits are of longer size (24). In higher metazoans, the ␣-subunits of CaM kinases II are especially abundant in brain (52). It remains to be tested whether, besides an induction of this gene, the resulting protein is activated by phosphorylation, as it is known to be for CaM kinases II from higher Metazoa (53). The results presented here identify an ethylene-mediated Ca 2ϩ -triggering signaling cascade in which the sponge CaM kinase II might be involved. In mammalian cells it has been established that Ca 2ϩ mediates also the CaM kinase II cascade and thereby prevents apoptosis (54). CaM kinases II are known to be involved in signal transduction reactions, mediating signal transmission or metabolic activity, and also in gene regulation (55). Here we show that the expression of CaM kinase II parallels the expression of the ethylene-responsive protein; future studies must show whether the expression of the latter gene is connected with a phosphorylation process mediated by CaM kinase II.
It should be stressed at this point that the CaM kinase II from S. domuncula is the phylogenetically oldest kinase of this family. Hence this enzyme represents a further autapomorphic character and a novel signal transduction molecule of Metazoa, such as the receptor tyrosine kinases (56) or integrin (26), which have been described previously from sponges. This study establishes that sponge cells react to ethylene with an activation of cell metabolism including gene activation. It will be interesting to perform studies of specific biological effects, such as the determination of ethylene on the development of sponge cells in primmorphs, or on the growth of bacteria in situ, which usually live in symbiosis with sponges (57).