Sponges (phylum Porifera) represent the phylogenetically oldest metazoan animals. Recently, from the marine sponge Geodia cydonium a first cDNA encoding a putative integrin receptor molecule was isolated. In the present study basic functional experiments have been conducted to test the hypothesis that in sponges integrin polypeptides also function as adhesion molecules and as outside-in signaling molecules. The sponge Suberites domuncula has been used for the experiments because from this sponge only has a cell culture been established. Here we report that aggregation factor (AF)-mediated cell-cell adhesion is blocked by the RGDS peptide which is known to interact with b integrin. Both RGDS and AF were found to stimulate DNA synthesis within 24 h. The b subunit of the integrin receptor was cloned from S. domuncula; the estimated 91-kDa molecule comprises the characteristic signatures. Evolutionary conservation of the b integrin was assessed by comparison with corresponding b integrin subunits from evolutionary higher metazoan taxa. Addition of RGDS or of AF to isolated cells of S. domuncula causes a rapid (within 1±2 min) increase in the intracellular Ca 2+ concentration which is further augmented in the presence of Ca 2+ . Furthermore, incubation of the cells with RGDS or AF causes an activation of the GTP-binding protein Ras. In addition it is shown that after a prolonged incubation of the cells with RGDS and AF the expression of the genes coding for Ras and for calmodulin is upregulated. These results suggest that the integrin receptor functions in the sponge system not only as adhesion molecule but also as a molecule involved in outside-in signaling.Keywords: Suberites domuncula, integrin, calcium, Ras, calmodulin, signal transduction, evolution, RGD(S), aggregation factor. Multicellularity in eukaryotes developed several times in evolution. Striking examples are the transition of unicellular to multicellular species within the volvocine algae (reviewed in [1]) which happened only 50±75 million years ago [2] while that of unicellular to multicellular animals dates back to 600 million years ago [3]. Until recently it remained unsolved if the evolution to multicellularity in Metazoa occurred only once (monophyly) or several times (polyphyly). Studies concentrating on those molecules which are considered to be characteristic for metazoan cells revealed that protein sequences, deduced from cDNAs obtained from the lowest metazoan phylum, the Porifera (sponges), are highly similar to those found in other metazoan phyla.Metazoan cells interact with each other differently than unicellular eukaryotes, especially with respect to (a) G-protein linked transmembrane receptors, (b) transmembrane adhesion molecules and (c) ligand-gated ion channels. Sequence data studies revealed that sponges ± the experiments have been performed mainly with the marine species Geodia cydonium and Suberites domuncula (reviewed in [4]) ± are provided with the G-protein linked transmembrane receptor, the metabotropic glutamate receptor [4a], ...

Among Metazoa, sponges (phylum Porifera) are the richest source for different bioactive compounds. The availability of the raw material is, however, restricted. To obtain enough of the bioactive compounds for application in human therapy, sponges have to be cultured in in vitro systems. One technique for the establishment of a long-term cell culture from sponges has recently been elaborated. Here, we present a procedure to cultivate tissue samples from sponges in an open system. The species Geodia cydonium, which produces bioactive compounds, has been selected. Tissue samples of approximately 10 g were attached to the bottoms of cultivation trays. After 2 to 3 days, the tissue samples formed a robust contact with the metal support. Subsequently, sets of trays, called tray batteries, either remained in huge aquaria at the Center for Marine Research or were transferred to the vicinity of a fish and mussel farm. The growth rates of the samples remained unchanged within the first month; however, after 3 and 6 months, they increased to 147% and 189%, respectively. In parallel, extracts were prepared from the tissue samples and tested for cytotoxicity in a mouse lymphoma cell assay system. Extracts from cultured tissue initially had a low inhibitory potency; however, after cultivation for 3 or 6 months, values comparable to those of extracts from sponges taken from the biotope were found. In addition, a molecular marker was applied to document the response (health state) of the tissue and the identity of the material in culture. The CD63 molecule was chosen because the expression of this molecule in mammalian systems changes with the age of the animals. The corresponding complementary DNA was isolated from Geodia cydonium. With this probe, the level of expression in cultured tissue samples decreased immediately after starting cultivation; after a cultivation period of 6 months, however, values were similar to those found in controls. These data show that sponge species that produce bioactive compounds can be cultivated in open systems, in which they retain their potency to produce bioactive compounds as well as their health state.

Recently cDNAs coding for cell surface molecules have been isolated from sponges. The molecules for a-integrin, galectin, and receptor tyrosine kinase (RTK), obtained from the marine sponge, Geodia c-vdonium, have been described earlier. In the present study also the cDNA for one putative P-integrin has been identified from G. cydonium. The deduced aa sequence comprises the characteristic signatures, found in other metazoan /3-integrin molecules; the estimated size is 95,215 Da. To obtain first insights into the molecular events which proceed during autograft fusion, the expressions of these genes were determined on transcriptional and translational level. The cDNAs as well as antibodies raised against the recombinant sponge proteins a-integrin, RTK and galectin were used and Northern blot experiments and immunocytochemical analyses have been performed. The results show that transcription of the two subunits of an integrin receptor as well as of the RTK are strongly upregulated after grafting; levels of > 10-fold have been determined in the fusion zone of the grafts after a 10 days incubation. Immunofluorescence studies of sections through the fusion zone support these data. In contrast the transcription of the gene encoding galectin is drastically downregulated after grafting. In a parallel series of experiments the level of the heat-shock protein-70 was determined and it was found that it remained unchanged after grafting. We conclude that integrin subunits and the RTK molecule are involved in self-self recognition of sponge.